JP2006158252A - Heat-resistant laccase and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a structurally stable laccase which is required in an industrial use, a DNA encoding the laccase, a recombinant vector including the gene DNA, a transformant transformed by the recombinant vector, and a method for producing a heat resistant laccase using the transformant. <P>SOLUTION: The laccase has following characteristics: (1) activity: the laccase strongly acts on a phenolic compound or heterocyclic compound in the presence of copper ions, (2): the optimum reacting temperature: 92°C under a condition of pH 5.0 and 10 min of the reaction time, (3) thermal resistance: the reduction of the enzymic activity are not found until 85°C when holding at pH 6.0 and 10 min, (4) half life at high temperature: the half life of the enzymic activity in holding at 85°C, pH 6.0 is more than 14 hrs, and (5) the molecular weight obtained by SDS-PAGE is 53 kDa. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to the production of a novel oxidase. More specifically, a novel laccase derived from a microorganism belonging to the genus Thermus having laccase activity, a gene encoding the enzyme, a recombinant vector containing the gene DNA, a transformant transformed with the recombinant vector, The present invention relates to a method for producing laccase using the transformant and a method for producing laccase using a cell-free synthesis system.

  Conventionally, laccase and polyphenol oxidase (hereinafter abbreviated as oxidase) are known as enzymes that oxidize aromatic compounds and heterocyclic compounds using molecular oxygen. Oxidase is widely distributed in microorganisms such as filamentous fungi, basidiomycetes and bacteria.

  In addition, among low-molecular-weight chemical substances that can become relatively stable reaction intermediate radicals among substrates that are oxidized by oxidases, particularly laccases, they are called laccase mediators or redox mediators. It causes phenomena such as enhancement of the oxidation reaction of laccase and oxidation of non-substrate chemical substances of laccase. As an example, it has been clarified that laccase can be indirectly oxidized in the presence of a redox mediator of a group of hardly biodegradable organic pollutant chemical substances that are difficult to decompose or cannot be decomposed by laccase alone (Non-Patent Document 1 and Non-Patent Document 1). (See Patent Document 2).

  Thus, it has been found that laccase has a catalytic ability for various chemical reactions resulting from the radical species of the reaction intermediate produced by the oxidation of the substrate. Therefore, the development of applications utilizing this catalytic ability is being actively continued by researchers all over the world. Examples of its use include dyeing and discharging, prevention of color transfer during washing, bleaching of pulp and fibers, decolorization of colored waste liquid, decomposition of refractory biodegradable compounds, detoxification of toxic chemicals, removal of bitter taste of food , Disappearance of moldy odor from wine cork, prevention of turbidity of juice, promotion of digestion of livestock feed, manufacture of non-adhesive wood board, manufacture of phenolic resin, manufacture of artificial lacquer paint, manufacture of adhesive, desulfurization of fossil fuel, Wide range of clinical analysis based on luminescence and color development, biosensor, synthesis of organic compounds, etc.

When laccase is used for the above-mentioned purposes, it is strongly desired that the laccase can be structurally stable and efficiently produced. However, many of the conventionally known laccases are unstable. For example, the laccase shown in Patent Document 1 has an optimum temperature of 50 ° C., can withstand heat treatment at 60 ° C. for 30 minutes, but is deactivated at higher temperatures. The heat-resistant laccase shown in Patent Document 2 is also stable up to 70 ° C when held at pH 7.0 for 10 minutes, but the optimum temperature is only 50 ° C. The most stable laccase known hitherto is an enzyme derived from Bacillus butyls, and the half-life of activity at 80 ° C. is about 112 minutes (see Non-Patent Document 3). However, the optimum enzyme temperature derived from Bacillus butyls is about 75 ° C., and when it is produced as a recombinant enzyme, most of it is insolubilized and it is difficult to mass-produce the active enzyme. Therefore, it is necessary to search and develop a new laccase in order to easily mass-produce laccase that works efficiently under higher temperature conditions.
JP 2004-208503 A JP 2002-171968 A Biotechnol. Lett. 22, 119-125 (2000) J. Biotechnol. 81,179-188 (2000) J. Biol. Chem. 277, 18849-18859 (2002)

  In view of the above, an object of the present invention is to produce a structurally stable laccase required for industrial use, a DNA encoding the laccase from which it is produced, and a recombinant vector containing the gene DNA Another object is to provide a transformant transformed with the recombinant vector, and a method for producing a thermostable laccase using the transformant.

  As a result of intensive studies to solve the above-mentioned problems, the inventor has cultivated microorganisms belonging to the genus hyperthermophilic Thermus, which is a source of many thermostable enzymes, and has laccase activity in the cells. Elucidation of the headline, its production, isolation and purification methods and properties, confirming that the enzyme is a laccase that is structurally stable and reacts efficiently under high temperature conditions. A large amount was expressed in the soluble fraction with Shelicia collie, laccase activity was confirmed, and the present invention was completed.

That is, the present invention provides the following aspects.
[1] Laccase with the following characteristics:
(1) Activity: strongly acts on phenolic compounds and heterocyclic compounds in the presence of copper ions; (2) Optimum reaction temperature: 92 ° C with a reaction time of 10 minutes at pH 5.0;
(3) Heat resistance: No decrease in enzyme activity up to 85 ° C when held at pH 6.0 for 10 minutes;
(4) Half-life at high temperature: when held at 85 ° C. and pH 6.0, the half-life of inactivation of enzyme activity is about 14 hours or more;
(5) The molecular weight measured by SDS-PAGE is 53 kDa.
[2] The laccase of [1] obtained by culturing a bacterium belonging to the genus Thermus.
[3] The laccase of [2], wherein the microorganism belonging to the genus Thermus is Thermus thermophilus HB27 strain.

[4] The following laccase protein (a) or (b):
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity
[5] The laccase protein according to [4], wherein the first glutamine of the amino acid sequence is pyroglutamylated.
[6] A nucleic acid encoding the following protein (a) or (b).
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity
[7] The following nucleic acid (c) or (d):
(c) a nucleic acid comprising the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing
(d) a nucleic acid capable of hybridizing under stringent conditions with a complementary strand of the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing and encoding a polypeptide having laccase activity

[8] The method for producing a laccase according to any one of [1] to [5], comprising culturing a microorganism belonging to the genus Thermus and recovering the laccase from the culture.
[9] An expression vector comprising the nucleic acid according to [6] or [7].
[10] A transformant obtained by transforming a host cell using the expression vector according to [9].
[11] A method for producing laccase, comprising culturing the transformant according to [10] and recovering laccase from the culture.
[12] A method for producing laccase, comprising producing a protein by a cell-free protein synthesis system using the nucleic acid according to [6] or [7].
[13] A laccase produced by the method of [11].
[14] A laccase produced by the method of [12].

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the efficient heat-resistant laccase by the genetic engineering method using the gene which codes the gene which codes the heat-resistant laccase which has the heat resistance laccase which is not large conventionally is provided. The laccase of the present invention has a high heat resistance so that no decrease in activity is observed even at 85 ° C. for 10 minutes, the optimum temperature is as high as 92 ° C., can maintain stability even in industrial use, and has a wide range Can be used for applications.

The heat-resistant laccase of the present invention is produced by a microorganism belonging to the genus Thermus. Examples of microorganisms belonging to the genus Thermus include Thermus thermophilus. Preferably, the Thermus thermophilus HB27 strain (DSM 703, ATCC BAA-163
) Can be used. The Thermus thermophilus HB27 strain can be obtained from ATCC (American Type Culture Collection).

  The laccase of the present invention differs from the conventionally known laccase not only in terms of amino acid sequence, but also has an optimum reaction temperature of 92 ° C., and there is no decrease in activity even after heating at 85 ° C. for 10 minutes. It is a novel enzyme different from laccase.

In the present invention,
(1) Laccase that reacts efficiently under high temperature conditions,
(2) Includes laccase with high heat resistance.

The laccase of the present invention is
(1) Activity: Strongly acts on phenolic compounds and heterocyclic compounds in the presence of copper ions;
(2) Optimal reaction temperature: 92 ° C with a reaction time of 10 minutes at pH 5.0;
(3) Heat resistance: No decrease in enzyme activity up to 85 ° C when held at pH 6.0 for 10 minutes;
(4) Half-life at high temperature: When kept at 85 ° C and pH 6.0, the half-life of inactivation of enzyme activity is about 14
Over 5 hours; and (5) 53 kDa molecular weight measured by SDS-PAGE
It has the characteristic. The molecular weight measured by MALDI-TOF MS is about 49 kDa. In addition, it catalyzes an oxidation reaction using ABTS, guaiacol, 2,6-dimethoxyphenol or the like as a substrate. Furthermore, the catalytic activity of the laccase of the present invention requires the presence of copper ions, and the concentration of copper ions is 10 μM or more, preferably 50 μM or more, more preferably 100 μM or more.

The laccase is a microorganism belonging to the genus Thermus, preferably the Thermus thermophilus HB27 strain under appropriate culture conditions (for example, a medium containing copper sulfate, for example, 72 ° C in Castenholz TYE medium.
Cell culture extract), and then a bacterial cell extract can be prepared and purified by appropriately combining ion exchange chromatography, hydrophobic chromatography, hydroxyapatite and the like. The Thermus thermophilus HB27 strain is described in, for example, Koyama Y, et al .. J. Bacteriol. 166: 338-340, 1986.

For example, Hitrap SP (Amersham Biosciences) is used as ion exchange chromatography, Butyl Toyopearl (Tosoh Corporation) is used as hydrophobic chromatography,
As hydroxyapatite, Toyopearl HA (manufactured by Tosoh Corporation) can be used.

Furthermore, the present invention is the following laccase protein (a) or (b).
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity.

Here, “one or several amino acids are deleted, substituted or added” is not limited, but is preferably 1 to 9, more preferably 1 to 5, most preferably 1 to 3 amino acids. Is deleted, substituted or added. When the amino acid sequence represented by SEQ ID NO: 4 is calculated using the amino acid sequence of SEQ ID NO: 4 and BLAST or the like as an amino acid sequence in which one or several amino acids are deleted, substituted or added (for example, BLAST default or initial condition parameters), having at least 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97%, 98% or 99% or more homology Are listed. The protein consisting of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 is substantially the same as the protein consisting of the amino acid sequence of SEQ ID NO: 4. Addition, deletion or substitution of amino acids is a well-known technique, for example, site-specific mutation described in Nucleic Acids Res. 10, 6487-6500 (1982) or random described in Technique, 1, 11-15 (1989). Can be performed by mutation.

For laccase activity, the sample whose activity is to be measured is reacted at 70 ° C in a solution containing 20 mM sodium acetate buffer (pH 5.0), 1 mM ABTS, 0.1 mM copper sulfate, and the change in absorbance at 418 nm is measured. Can be measured.

  Furthermore, the present invention also includes a laccase protein obtained by pyroglutamylating the first glutamine of the amino acid sequence in the protein (a) or (b).

Furthermore, the present invention is a nucleic acid encoding the following protein (a) or (b).
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity.

Here, the nucleic acid includes DNA and RNA.
Furthermore, the present invention provides the following nucleic acid (c) or (d).
(c) a nucleic acid comprising the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing.
(d) a nucleic acid capable of hybridizing under stringent conditions with a complementary strand of the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing and encoding a polypeptide having laccase activity.

  Here, the stringent conditions may be known stringent conditions. For example, low ionic strength, high temperature washing conditions such as 0.015 M sodium chloride, 0.0015 M sodium citrate, 0.1% sodium lauryl sulfate. The conditions for washing at 50 ° C. can be mentioned.

The nucleic acid of the present invention can be obtained by a known genetic engineering technique. For example, it can be performed according to the description of Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell, DW. Cold Spring Harbor Laboratory Press edited by Sambrook et al.
For example, it can be obtained by extracting mRNA from the Thermus thermophilus HB27 strain, preparing a cDNA library using an appropriate host, and screening using laccase activity as an indicator. In addition, design and synthesis of an appropriate probe from the DNA sequence obtained from the determined amino acid sequence,
Laccase genes can also be obtained by cloning. Furthermore, an appropriate primer can be designed from the nucleic acid sequence, and the laccase gene can be amplified by a gene amplification method such as PCR. Furthermore, it can be obtained by chemical synthesis according to the DNA sequence obtained from the amino acid sequence of the laccase.

Furthermore, the present invention includes a recombinant vector containing the above nucleic acid DNA.
For example, pET32 (a) +, pET29a (+), pUC18, pUC118, etc. can be used as a vector for expression of the thermostable laccase gene when Escherichia coli is used as the host microorganism.
Of course, the nucleic acid sequence of the laccase gene of the present invention can be incorporated into a recombinant vector after being changed in accordance with the codon usage frequency in the host. The expression vector in the present invention includes a structure in which the gene (that is, laccase gene) is linked downstream of a promoter capable of expressing a gene encoding a protein having laccase activity. Can be produced by linking (inserting). Those having a structure in which a laccase gene is linked downstream of a selection marker for Neisseria gonorrhoeae and Escherichia coli and a promoter for expressing the laccase gene are preferable. Further, the laccase gene in the present invention needs to be incorporated into a vector so that its function is exhibited. Therefore, in addition to the promoter and the DNA of the present invention, a terminator and a ribosome binding sequence may be incorporated into the vector of the present invention. Such operations are usually performed in the art, and can be appropriately performed by those skilled in the art.

The present invention also includes a transformant transformed with a recombinant vector containing the above DNA.
As the host, microorganisms, insect cells, animal cells, or plant cells can be used, and examples thereof include Escherichia coli.

  As a means for introducing the recombinant vector into the host, a known method suitable for each host can be used, and examples thereof include a calcium chloride method, an electroporation method, and a high-temperature shock method.

Furthermore, the present invention provides a method for producing a heat-resistant laccase, comprising the steps of: culturing the transformant in a medium; and recovering laccase from the microbial cells. A method for producing a thermostable laccase characterized by producing a laccase by a cell-free protein synthesis system containing DNA to be produced, and a laccase obtained by the method are also included. The transformant of the present invention is cultured by a usual method used for culturing a host. Various culture methods are known for all types of host cells, and those skilled in the art can easily select an appropriate method. After cultivation, the recombinant protein of the present invention is collected from the culture. When the protein is produced outside the cells or extracellularly, the enzyme can be collected using the culture solution as it is or after removing the cells by centrifugation. When the protein is in a cell, the enzyme may be collected after disrupting the cell by various known methods to obtain a cell suspension. The enzyme may be collected by using a general biochemical method used for protein isolation and purification, for example, ammonium sulfate precipitation, affinity chromatography, ion exchange chromatography, etc. alone or in appropriate combination. it can.

Cell-free protein synthesis is performed in vitro, from E. coli and wheat germ to ribosome, aminoacyl-tRNA synthetase, translation initiation factor, translation elongation factor, translation termination factor, etc. Cell-free translation extract (S30 extract) containing ATP, ATP regeneration system, promoter and plasmid containing nucleic acid encoding laccase of the present invention or mRNA encoding the laccase, tRNA, RNA polymerase, RNAase inhibitor, ATP , GTP, CTP, UTP and other energy sources, buffer agents, amino acids, salts, antibacterial agents and the like are mixed, and the laccase is translationally synthesized using the DNA encoding the laccase of the present invention as a template. The plasmid to be used is not limited, and a known plasmid can be used. It can be used by introducing an appropriate promoter, ribosome binding site, etc. by a known genetic engineering technique. A person skilled in the art can appropriately select a plasmid used in the present invention and design and construct it by himself. The ATP regeneration system is not limited, and a known combination of phosphate donor and kinase can be used. Examples of this combination include phosphoenolpyruvate (PEP) -pyruvate kinase (PK), creatine phosphate (CP) -creatine kinase (CK), acetyl phosphate (AP) -acetate kinase (AK) Combinations may be mentioned, and these combinations may be added to the cell-free protein synthesis system. As the promoter, an endogenous promoter possessed by the microorganism used in the cell-free protein synthesis system of the present invention may be used, or an exogenous promoter may be used. When an exogenous promoter is used and the endogenous polymerase of the microorganism used in the cell-free protein synthesis system cannot be transcribed, RNA polymerase that acts on the promoter is added to the system of the present invention. For example, when a T7 promoter is used as a promoter, T7 RNA polymerase is added. As the promoter, Trc promoter, T7 promoter, Tac promoter, etc. can be used. Examples of the buffer include Hepes-KOH and Tris-Acetate, and examples of the salts include acetate, glutamate and the like, but are not limited thereto. What is necessary is just to determine each density | concentration suitably. For example, JP 2000-514298, JP 2000-175695, JP 2002-338597, Zubay, Annu. Rev. Genet. 7 267-287 [1973], Pratt, Transcription and Translation-a practical approach, Henes, BD and Higgins, SJ ed., IRL Press, Oxford. 179-209 [1984], Kim et al., Eur. J. Biochem. 239 881-886 [1996], Nakano et al., Biosci. Biotechnol. Biochem. 58 631 -634 [1994], Madin et al., Proc. Natl. Acad. Sci. USA 97 559-564 [2000]) and the like.

It can also be performed by the flow method. The flow method means that protein synthesis is performed by continuously supplying ATP, GTP, etc., which are easily depleted from the reaction system, with a pump, etc. during protein synthesis, and removing reaction byproducts that inhibit protein synthesis. This is a method for a long time. The flow method can be performed by the method described in Spirin et al., Science 242 1162-1164 [1988]), Kim and Choi, Biotechnol. Prog. 12 645-649, and the like.

Cell-free synthesis can also be performed using a commercially available cell-free synthesis kit or device. As a commercially available kit or device, PROTEIOS ^TM cell-free protein synthesis kit manufactured by Toyobo Co., Ltd., Post Genome Institute, Inc. PURESYSTEM (registered trademark) manufactured by Roche Diagnostics, Inc. and RTS Proteo Master manufactured by Roche Diagnostics.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

[Example 1] Culture of thermus thermophilus and detection of laccase 8 g of tryptone, 4 g of yeast extract and 2 g of sodium chloride were added to 1 L of distilled water, and the pH was adjusted to 7.0 with sodium hydroxide ( The following is abbreviated as TYM medium), heat-sterilized by autoclave, inoculated with Thermos thermophilus HB27, and stirred at 100 rpm for 72 ° C.
And stirred for 20 hours. The main culture was added to a fresh medium in which copper sulfate was added to the TYM medium to a final concentration of 0, 0.1, 0.5, or 1 mM, and cultured at 72 ° C. for 20 hours with stirring. Centrifugation (5,000 g
10 minutes), and a bagbuster solution made by Novagen and benzonase made by Novagen were added to the precipitate and suspended. The cell suspension is stirred at room temperature for 20 minutes and centrifuged (20,000 g
, 20 minutes). Extract this bacterial cell extract from 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, 1 mM 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (hereinafter abbreviated as ABTS) As shown in Fig. 1, green color was seen in the reaction system using the bacterial cell extract prepared from the medium added with copper sulfate, and laccase activity was confirmed. It was. No activity was detected from the bacterial cell extract cultured in a medium not containing copper sulfate, and it was found that laccase activity was induced as the concentration of copper sulfate increased.

[Example 2] Purification of laccase From the results obtained in Example 1, using 250 mL of TYM medium containing 1 mM copper sulfate, [Example 1]
Thermus thermophilus was cultured according to the described method to prepare a crude enzyme extract. This extract was purified by cation exchange column chromatography (Hi-Trap SP, Amersham Biosciences). The column was equilibrated with 20 mM Tris-HCl buffer (pH 7.0), and the crude enzyme extract was added. When the non-adsorbed fraction was washed with 20 mM Tris-HCl buffer (pH 7.0), ABTS oxidation activity was not detected. Next, the adsorbed protein was eluted with a linear concentration gradient of 0-0.2 M sodium chloride, and the ABTS oxidation activity fraction was recovered.

Next, it was separated by hydrophobic column chromatography (Butyl Toyopearl, manufactured by Tosoh Corporation). Ammonium sulfate was added to the active fraction eluted from the cation exchange column to a final concentration of 1.0 M and adsorbed on a column equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 1.0 M ammonium sulfate. When the non-adsorbed fraction was washed with 20 mM Tris-HCl buffer (pH 7.0) containing 1.0 M ammonium sulfate, ABTS oxidation activity was not detected. Next, the adsorbed protein was eluted with a linear gradient of 1.0-0 M ammonium sulfate, and the ABTS oxidation activity fraction was recovered.

[Example 3] Electrophoretic analysis of laccase About 0.5 μg of the sample obtained in Example 2 was subjected to SDS-polyacrylamide gel (10%) electrophoresis according to a conventional method. After the electrophoresis, the gel was rinsed lightly with water and stained with Coomassie Brilliant Blue according to a conventional method. A band consisting mainly of a molecular weight of about 53 kDa was detected (Figure 2). Since this band became darker as the degree of purification improved, it was estimated that this band was laccase.

[Example 4] Analysis of amino terminus of laccase Next, in the same manner as in Example 3, SDS-polyacrylamide gel was analyzed under the same conditions as in Example 3 for the purpose of analyzing the amino terminus of about 2 μg of the sample obtained in Example 2. (10%) Subjected to electrophoresis. After the gel was lightly rinsed with 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid buffer (pH 9.0), the protein was electrically transferred according to a conventional method to a Millipore Immobilon membrane hydrophilized with methanol. A 0.1 M N-cyclohexyl-3-aminopropanesulfonic acid buffer (pH 9.0) was used as the transfer buffer. Next, the immobilon membrane was stained with Coomassie brilliant blue, and a stained band corresponding to a molecular weight of about 53 kDa was cut out. When this sample was subjected to the Procise 494 cLC system manufactured by Applied Biosystems and analyzed for the amino terminal sequence, no specific sequence was detected. As a result, it was found that the amino terminal of laccase purified from the Thermus thermophilus cells was blocked.

[Example 5] Mass spectrometry and mass-fit fingerprinting search of tryptic digestion fragment of laccase Next, a band with a molecular weight of about 53 kDa detected in Example 3 was cut out with a sharp cutter knife. This sample was trypsin-degraded by in-gel digestion according to a conventional method, and subjected to mass spectrometry by MALDI-TOF using Voyager-DE STR manufactured by Applied Biosystems. As a result of mass-fit fingerprinting search for a known amino acid sequence database using the identified 121 mass data, 36 peptides were hit against the deduced amino acid sequence (SEQ ID NO: 1) derived from Thermus thermophilus HB27 did. The function of the sequence shown in SEQ ID NO: 1 is presumed to be a laccase-like protein from homology with other gene sequences. However, it is actually purified as a protein and its enzymatic activity has not been experimentally confirmed. Further, the sequence shown in SEQ ID NO: 1 shows only 40% or less amino acid sequence homology with known laccases whose properties have been purified and whose properties have been clarified. At such low values, it is impossible to determine the function of a protein from sequence homology. Furthermore, it is impossible to estimate from the amino acid sequence over the heat resistance. As is clear from the results of Example 4, the amino terminus of the purified laccase is blocked and does not start with methionine as shown in SEQ ID NO: 1.

[Example 6] Prediction of amino terminus and entire sequence of laccase Since the amino terminus of the purified laccase activity was blocked, the amino acid residue corresponding to the amino terminus of the enzyme, the blocked properties, etc. were clarified. Absent. Therefore, the presence or absence of a signal peptide was predicted for SEQ ID NO: 1 using gene analysis software (Genetyx). As a result, it was strongly predicted that the peptide bond between the 22nd and 23rd amino acids of SEQ ID NO: 1 was cleaved by signal peptidase. Based on this result, as a result of comparison with the detailed mass spectrometry result of the trypsin fragment obtained in Example 5, the signal detected at m / z 969.4557 was 9th from the 23rd to the 31st of SEQ ID NO: 1. The peptide consisted of an amino acid, which coincided with the one obtained by pyroglutamylation at the 23rd residue (pyroglutamic acid). According to the sequence shown in SEQ ID NO: 1, the 22nd residue is alanine, and the sequence consisting of alanine-glutamine is not subject to trypsin digestion. Therefore, it was concluded that the peptide bond between the 22nd and 23rd was cleaved with signal peptidase and the terminal glutamine was pyroglutamylated, as predicted by genetic analysis software.

  The signal detected at m / z 2566.1890 was consistent with the mass of the 22-residue sequence at the carboxyl terminus of SEQ ID NO: 1. As a result, it was found that laccase was a sequence corresponding to SEQ ID NO: 4 composed of the 23rd to 462th sequences among the sequences of SEQ ID NO: 1.

[Example 7] Cloning of laccase gene Next, the inventor decided to clone a gene to which a methionine codon was added as the start codon of SEQ ID NO: 4. Gene cloning was performed by polymerase chain reaction using the chromosomal DNA of Thermus thermophilus strain HB27 as a template.

(1) Preparation of chromosomal DNA Chromosomal DNA of Thermus thermophilus HB27 (DSM 703, ATCC BAA-163) was prepared by the following method. The strain was cultured in 200 mL of TYM medium with shaking at 70 ° C. overnight, and then collected by centrifugation (5,000 g, 10 minutes). The cells were washed once with 0.15 M sodium chloride and 0.1 M ethylenediaminetetraacetic acid, and collected again by centrifugation (5,000 g, 10 minutes). Part of the cells (0.6 g) was suspended in a solution containing 12 mL Tris-HCl buffer (pH 9.0), 1% sodium lauryl sulfate, and 0.1 M sodium chloride, and freeze-thaw was repeated twice to lyse. 1 mg of proteinase K was added to this solution, and the mixture was incubated at 50 ° C. for 3 hours. Thereafter, an equal amount of phenol was added, the mixture was slowly stirred, the aqueous layer and the oil layer were separated by centrifugation (5,000 g, 10 minutes), and the aqueous layer was recovered. This operation was further repeated twice, and twice the amount of ethanol was gently added to the finally collected aqueous layer. The precipitated DNA was wound up with a glass rod, washed with 70% ethanol, and dissolved in 2 mL of 10 mM Tris-HCl buffer and 1 mM ethylenediaminetetraacetic acid.

(2) A DNA fragment containing a gene encoding laccase and amplification of the DNA fragment From the DNA sequence of the putative laccase gene found in Example 6, an oligonucleotide primer for polymerase chain reaction was synthesized. The sequence is upstream: 5′-CATATGCAAGGCCCTTCCTTCCCC-3 ′ (SEQ ID NO: 2), downstream: 5′-AAGCTTAACCCACCTCGAGGACTCC-3 ′ (SEQ ID NO: 3). The upstream sequence is designed to add a codon encoding methionine as a start codon to the amino acid sequence of SEQ ID NO: 4 and include the restriction enzyme site NdeI, and the downstream sequence inserts a stop codon after the amino acid at the carboxyl end, It was designed to include the restriction enzyme site HindIII. An amplification reaction was carried out using these both-end primers, using genomic DNA derived from Thermus thermophilus as a template, and KOD-Plus-DNA polymerase (manufactured by Toyobo Co., Ltd.) as the polymerase. Amplification was performed by keeping the reaction solution at 94 ° C. for 2 minutes and then repeating a cycle consisting of 94 ° C.-15 seconds, 55 ° C.-30 seconds, 68 ° C.-1 minutes 25 times.

(3) Construction of laccase expression plasmid The PCR product consisting of about 1,000 base pairs obtained in (2) was separated by agarose gel electrophoresis according to a conventional method, purified, and then the PCR ^R- Blunt II-TOPO ^R cloning kit ( Invitrogen) was used for cloning. Part of the reaction solution was introduced into an Escherichia coli JM109 competent cell (Toyobo Co., Ltd.) and placed on an LB (1% tryptone, 0.5% yeast extract, 1% sodium chloride) agar plate containing 50 μg / mL kanamycin. Plasmids were prepared from growing colonies. Next, this recombinant plasmid was simultaneously cleaved with NdeI and HindIII, and the resulting approximately 1 kbp DNA fragment was previously digested with NdeI and HindIII to form a linear plasmid of pET32a (+) manufactured by Novagen. The ligation reaction was performed by ligation according to the usual method. A part of the reaction solution was introduced into a competent cell of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.) and cultured on an LB agar plate containing 100 μg / mL ampicillin. A single colony was cultured, a plasmid was prepared according to a conventional method, and the recombinant plasmid was named pET32T27Lcs1.

[Example 8] Determination of base sequence Next, the base sequence of the insert region of the recombinant plasmid pET32T27Lcs1 obtained in Example 7 was determined. The sequencing reaction was performed by the dideoxy method using Big Dye version 3.1 kit manufactured by Applied Dubai Systems. The base sequence was analyzed using a DNA sequencer (Prism 310) manufactured by Applied Dubai Systems. The determined base sequence and deduced amino acid sequence are shown in SEQ ID NOs: 4 and 5 in the sequence listing.

[Example 9] Expression of recombinant laccase gene
pET32T27Lac1 and Takara Bio pGro7 were simultaneously introduced into Novagen Escherichia coli competent cell BL21 (DE3) on an LB agar plate containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol at 37 ° C. Incubated overnight. 100 single colonies
Inoculate 1 L of LB liquid medium containing μg / ml ampicillin, 34 μg / mL chloramphenicol, 0.2% arabinose, overnight express autoinduction kit 1 (Novagen), 100 times per minute at 30 ° C For 20 hours.

[Example 10] Purification of recombinant laccase (1) Purification by heat treatment The culture solution obtained in Example 9 was centrifuged to recover the cells. Next, the cells were dissolved in 40 mL of Novagen bugbuster solution, and 4 μL of Novagen Benzonase was further added. The cell suspension was stirred at room temperature for 20 minutes, and the precipitate was removed by centrifugation (5,000 g, 10 minutes). The supernatant was then heated in a 65 ° C. water bath for 15 minutes, and the precipitate was removed by centrifugation (20,000 g, 20 minutes). The supernatant was collected and the ABTS oxidation activity was measured. As a result, the same activity as that before the heat treatment was maintained, and the activity was not decreased by the heat treatment.

(2) Purification by cation exchange column chromatography Next, the supernatant after the heat treatment was purified by cation exchange column chromatography. High Trap SP (Amersham Bioscience) was equilibrated with 20 mM Tris-HCl buffer (pH 7.0), and the crude enzyme extract of the heat-treated supernatant was added. Next, the non-adsorbed fraction was washed with 10 volumes of 20 mM Tris-HCl buffer (pH 7.0). ABTS oxidation activity was not detected in this fraction. Next, the adsorbed protein was eluted with a linear concentration gradient of 0-0.2 M sodium chloride in a 20-fold volume of the column, and the ABTS oxidation activity fraction was recovered.

(3) Purification by hydrophobic column chromatography Next, the column was separated by hydrophobic column chromatography. Butyl Toyopearl (Tosoh Corp.) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) containing 1.0 M ammonium sulfate to the active fraction eluted from the cation exchange column to a final concentration of 1.0 M It was made to adsorb to. Next, when the non-adsorbed fraction was washed with 20 mM Tris-HCl buffer (pH 7.0) containing 10 volumes of 1.0 M ammonium sulfate, no ABTS oxidation activity was detected. Next, the adsorbed protein was eluted with a linear concentration gradient of 1.0-0 M ammonium sulfate in 20 volumes of the column, and the ABTS oxidation activity fraction was recovered.

(4) Purification by hydroxyapatite column chromatography Next, it was separated by hydroxyapatite column chromatography. The active fraction eluted from the cation exchange column was dialyzed against 20 mM sodium phosphate (pH 7.0) and adsorbed on Toyopearl HA (manufactured by Tosoh Corporation) equilibrated with 20 mM sodium phosphate (pH 7.0). When the non-adsorbed fraction was washed with 10 column volumes of 20 mM sodium phosphate (pH 7.0), ABTS oxidation activity was not detected. Next, the adsorbed protein was eluted with a linear concentration gradient of 20 mM-0.5 M sodium phosphate (pH 7.0) in a volume 20 times that of the column, and the ABTS oxidation activity fraction was recovered. This purified sample was used as the final sample in the following experiments.

[Example 11] Mass spectrometry of recombinant laccase Mass spectrometry of the recombinant laccase obtained in Example 10 was performed. The protein solution was desalted by ultrafiltration (molecular weight cut-off 10,000) using Ultra U-15 manufactured by Amicon and analyzed by a mass spectrometer Voyager-DE STR manufactured by Applied Systems. The mass was determined to be 48,990.87. As a result, the mass of the recombinant laccase coincided with the mass calculated from the amino acid sequence of SEQ ID NO: 4.

[Example 12] Analysis of amino terminus of recombinant laccase Furthermore, the recombinant laccase obtained in Example 10 was separated by electrophoresis and transferred to an immobilon membrane in the same manner as described in Example 4. When this sample was subjected to Procise 494 cLC system manufactured by Applied Biosystems and analyzed for the amino terminal sequence, a sequence corresponding to the first 5 residues of the amino acid sequence described in SEQ ID NO: 4 was obtained. As suggested from the results of Example 11, the recombinant laccase was found to be a protein consisting of the sequence shown in SEQ ID NO: 4.

[Example 13] Properties of recombinant laccase (1) Dependence of activity on copper ion concentration The activity of the enzyme was not expressed in the absence of copper ion. Therefore, the activity was measured in a reaction solution in which 0.05 μM-1.0 mM of copper ion concentration was added to an activity measurement solution containing 20 mM sodium acetate buffer (pH 5.0) and 1 mM ABTS. As a result, as shown in FIG. 3, the activity of this enzyme was dependent on the copper ion concentration, and the apparent binding constant was calculated to be 30.4 μM from the sigmoid curve approximation. In the following experiment, 0.1 mM copper sulfate, at which the copper ion concentration necessary for activity reached almost saturation, was added to the activity measurement solution.

(2) Substrate-specific activity The activity of this enzyme was measured using ABTS, syringaldazine, guaiacol, and 2,6-dimethoxyphenol known as laccase substrates. 0.8 μg of enzyme was added to an activity measurement solution in which 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate and 1 mM substrate were mixed, and the activity was measured at 70 ° C. for 1 minute. As a result, coloration accompanying oxidation was observed for any substrate. The color reaction was measured at 418 nm for ABTS, 525 nm for syringaldazine, 468 nm for guaiacol and 2,6-dimethoxyphenol, and the activity against each substrate was as shown in Table 1. However, ABTS was 36000 M-1 cm-1, syringaldazine was 65000 M-1 cm-1, guaiacol was 6400 M-1 cm-1, and 2,6-dimethoxyphenol was 49600 M-1 cm-1. The results are shown in the table below.

Substrate activity (μmol / min / mg protein)
ABTS 47
Syringaldazin 32
Guaiacol 29
2,6-dimethoxyphenol 108

(3) Temperature dependency of activity The temperature dependency of the activity of this enzyme was measured in the temperature range of 65 ° C to 99 ° C. Enzyme solution is added to an activity assay solution containing 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, and 1 mM ABTS, incubated at each temperature for 10 minutes, cooled in ice water, and absorbance at 418 nm. Was measured. As a result, as shown in FIG. 4, the enzyme was found to have an optimum temperature around 92 ° C. From the above, it was found that this enzyme reacts efficiently at high temperatures. Among conventionally known laccases, an enzyme derived from Bacillus butyris is known as an enzyme having high heat resistance, and the optimum temperature of the enzyme derived from Bacillus butyris is 75 ° C. (Non-patent Document 3). Therefore, the laccase of the present invention is superior in reactivity at a higher temperature than the enzyme derived from Bacillus butyris.

(4) pH dependence of activity The pH dependence of the activity of this enzyme was measured in the range of pH 2.5-9.0. In an activity assay solution containing 50 mM Britton & Robinson buffer (50 mM boric acid, 50 mM acetic acid, 50 mM phosphoric acid mixed buffer, pH adjusted with sodium hydroxide), 0.1 mM copper sulfate, 1 mM ABTS The enzyme solution was added to the solution, incubated at 70 ° C. for 10 minutes, cooled in ice water, and the absorbance at 418 nm was measured. As a result, as shown in FIG. 5, the enzyme was found to have an optimum pH around pH 4.5.

(5) Heat resistance
The enzyme solution dissolved in 20 mM sodium acetate buffer (pH 6.0) was heated at each temperature for 10 minutes and allowed to stand in ice water. The enzyme activity was measured in an activity measurement solution containing 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, and 1 mM ABTS. As a result, as shown in FIG. 6, this enzyme maintains the same level of activity as when not heat-treated at 85 ° C. or lower, and has an activity of 90% or higher at 90 ° C. and 66% at 100 ° C. It was. The thermostable laccase shown in Patent Document 1 is stable only up to 70 ° C., and the thermophilic bacterium enzyme has much higher thermostability.

(6) Half-life of activity at high temperature
The enzyme solution dissolved in 20 mM sodium acetate buffer (pH 6.0) was kept warm at 80 ° C., and a part of the enzyme solution was taken at regular intervals and left in ice water. The enzyme activity was measured in an activity measurement solution containing 20 mM sodium acetate buffer (pH 5.0), 0.1 mM copper sulfate, and 1 mM ABTS. As a result, a change with time of the activity as shown in FIG. 7 was obtained. From this graph, the half-life of this enzyme at 80 ° C. was calculated to be 868 minutes (14 hours or longer). Among conventionally known laccases, an enzyme derived from Bacillus butyris is known as an enzyme having high heat resistance. It is known that an enzyme derived from Bacillus butyris exhibits a half-life of 112 minutes at 80 ° C. (J. Biol. Chem. 277, 18849-18859 (2002)). The laccase of the present invention is an enzyme derived from Bacillus butyris. It had much higher heat resistance than that.

It is a figure which shows the effect which the density | concentration of the copper sulfate added to a thermus thermophilus culture medium has on laccase activity. It is a figure which shows the result of the SDS-polyacrylamide gel (10%) electrophoretic analysis of the laccase refine | purified from Thermus thermophilus. The left lane is a molecular weight marker, and the right lane is a purified laccase. It is a figure which shows the copper ion density | concentration dependence of the Thermus thermophilus origin laccase activity. It is a figure which shows the temperature dependence of the Thermus thermophilus origin laccase activity. It is a figure which shows the pH dependence of Thermus thermophilus origin laccase activity. It is a figure which shows the heat resistance of the thermus thermophilus origin laccase activity. It is a figure which shows the half life of the activity at the time of heat-retaining at 80 degreeC of the thermus thermophilus origin laccase.

  SEQ ID NOs: 2 and 3 primers

Claims (14)

Laccase with the following characteristics:
(1) Activity: strongly acts on phenolic compounds and heterocyclic compounds in the presence of copper ions; (2) Optimum reaction temperature: 92 ° C with a reaction time of 10 minutes at pH 5.0;
(3) Heat resistance: No decrease in enzyme activity up to 85 ° C when held at pH 6.0 for 10 minutes;
(4) Half-life at high temperature: when held at 85 ° C. and pH 6.0, the half-life of inactivation of enzyme activity is about 14 hours or more;
(5) The molecular weight measured by SDS-PAGE is 53 kDa.   The laccase according to claim 1, which is obtained by culturing a bacterium belonging to the genus Thermus.   The laccase according to claim 2, wherein the microorganism belonging to the genus Thermus is Thermus thermophilus HB27 strain. The following laccase protein (a) or (b):
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity   The laccase protein according to claim 4, wherein the first glutamine of the amino acid sequence is pyroglutamylated. A nucleic acid encoding the following protein (a) or (b):
(a) A laccase protein comprising the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing
(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing and having laccase activity The following nucleic acid (c) or (d):
(c) a nucleic acid comprising the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing
(d) a nucleic acid capable of hybridizing under stringent conditions with a complementary strand of the polynucleotide sequence represented by SEQ ID NO: 5 in the sequence listing and encoding a polypeptide having laccase activity   The method for producing laccase according to any one of claims 1 to 5, comprising culturing a microorganism belonging to the genus Thermus and recovering laccase from the culture.   An expression vector comprising the nucleic acid according to claim 6 or 7.   A transformant obtained by transforming a host cell with the expression vector according to claim 9.   A method for producing a laccase comprising culturing the transformant according to claim 10 and recovering the laccase from the culture.   A method for producing laccase, comprising producing a protein by a cell-free protein synthesis system using the nucleic acid according to claim 6 or 7.   A laccase produced by the method according to claim 11.   A laccase produced by the method according to claim 12.

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