JP2002253250A - Metallothionine gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extracellularly mass-produce a protein as a fused protein by cultivating a transformant. SOLUTION: Cloning of metallothionine gene is carried out and its base sequence is also determined. The new cloned gene and a glucoamylase gene fragment are inserted into a vector to transform a host (Aspergillus oryzae).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a metallothionein gene of Aspergillus oryzae. The present invention relates to a method for mass-producing metallothionein using a metallothionein gene newly isolated from Aspergillus oryzae. It is used for removal of water.

[0002] The present invention also relates to A. o using a metallothionein gene newly isolated from Aspergillus oryzae.
A fusion protein with glucoamylase of ryzae that is secreted and produced in large quantities using Aspergillus oryzae, and can be used in various bioremediation fields such as designing molecules suitable for biosolvents that can capture harmful heavy metals in the environment. It is.

[0003]

2. Description of the Related Art Metallothionein is a heavy metal binding protein widely present in various eukaryotes such as vertebrates, invertebrates, plants, yeasts, and molds. Metallothionein is Cd,
Heavy metals such as Zn, Cu, Hg, and Ag can be widely adsorbed, and the number of adsorbed molecules per molecule is large. Metallothionein is a low molecular weight protein with a molecular weight of about 3000 for Neurospora crassa and about 7000 for animals. This protein binds to various heavy metals such as zinc and cadmium in vivo, and is presumed to function as a heavy metal scavenger. Recently, new physiological functions have been elucidated, for example, the association between metallothionein and the development of cancer or Alzheimer has been pointed out, and it has been clarified that metallothionein regulates the immune system. As described above, metallothionein is a protein having a very important function involved in various control systems in vivo.

On the other hand, the excellent heavy metal binding ability of metallothionein is expected to be applied as a heavy metal adsorbent in the environment. Attempts are being made to remove environmental pollution due to mercury, which has become a problem with Minamata disease, using this metallothionein. 9 of metallothionein derived from Neurospora crassa
It has been reported that a dimer is prepared using a genetic engineering technique and a strain having a heavy metal adsorption capacity of 65 times that of a wild type strain is bred.

[0005] As described above, metallothionein is a protein having a high utility value in terms of environmental engineering, but applied research such as high production and environmental application of the present protein has hardly been performed.
In order to utilize metallothionein for bioremediation, it is essential to develop a protein having a high adsorptive capacity and to develop a technology for producing the protein in a high amount.

When a protein is produced by recombinant DNA technology, Escherichia coli is often used as a host for substance production. When the gene is expressed in Escherichia coli, the recombinant protein is produced as an intracellular protein. However, intracellular proteins require an operation of crushing the cells by ultrasonic treatment or the like, and there is a limit in improving productivity. Generally, it is said that the cost of an enzyme used industrially is not suitable for intracellular production. In order to efficiently express the isolated gene and produce an industrially usable protein, it is necessary to secrete the enzyme outside the cells. In addition, when an enzyme is produced in a host other than the organism from which the gene was isolated, it becomes a recombinant protein, and there is a problem that its use in foods and the like is limited.

On the other hand, Aspergillus oryzae, a koji mold, is a microorganism that has been used for sake brewing for many years and has a very high protein secretion ability. If a metallothionein gene having industrially useful activity is present in the koji mold, mass production with the koji mold is possible, and it is expected that both the productivity problem and the heterogeneous gene expression problem can be solved at once. Is done. In addition, aspergillus can be cultured in various ways from liquid culture to solid culture depending on the production of the enzyme, and the enzyme can be produced according to the industrial use.

[0008]

The present invention provides a new technique for efficiently producing a protein having a metallothionein-like heavy metal-capturing activity (including metallothionein itself) by using a genetic engineering technique. It was made for the purpose of development.

[0009]

Means for Solving the Problems The present invention has been made to achieve the above object, and as a result of earnest research,
Successful cloning of the gene encoding metallothionein and determination of its nucleotide sequence, further preparation of a transformant containing the gene, expression of the gene, and a protein having metallothionein or metallothionein-like activity by culturing the transformant Although metallothionein, which is useful for biosorbent, which is a microorganism-derived adsorbent used for removal and recovery of heavy metals in aqueous solution, is expressed in Escherichia coli, it is produced as intracellular protein, Problems arise in separation and the like. Therefore, in order to further improve this point, by conducting research and designing a fusion protein with a glucoamylase molecule derived from Aspergillus, we succeeded for the first time to produce a highly pure metallothionein fusion protein outside the cells with high secretion. ,
The present invention has been completed. Hereinafter, the present invention will be described in detail.

[0010] In order to clone the metallothionein gene, A. mRNA prepared from cells of oryzae, cDNA synthesized from this mRNA
Build a library. The nucleotide sequence of this library is comprehensively determined, and homology between each sequence and a known gene database is searched. From these cDNA clones, clones exhibiting high homology with the metallothionein gene of Neurospora crassa were searched, and from this clone A. oryzae metallothionein gene is isolated.

The isolated gene is a metallothionein gene, which is a gene encoding metallothionein, a gene encoding a protein having a metallothionein-like heavy metal-capturing activity, and at least one of these genes and another gene. It refers to at least one selected from a fusion gene and a gene containing at least one of these three types of genes (hereinafter sometimes simply referred to as a metallothionein gene). In the present invention, the metallothionein gene thus obtained is introduced into a host and expressed therein, and at least a metallothionein, a protein having a metallothionein-like heavy metal-capturing activity, or a fusion protein thereof with another protein. It is one that produces one.
In the present invention, the above three kinds of proteins include metallothionein.

As the host, various microorganisms can be used as appropriate, but the present invention is characterized in that eukaryotes can be used and the target enzyme can be efficiently produced in large quantities. Eukaryotes such as Aspergillus A. oryz
ae is a filamentous fungus used in the traditional fermentation industry in Japan, such as sake, soy sauce, and miso. A feature of this strain is that it produces very large quantities of proteins useful in the fermentation industry. Due to the high protein-producing ability of this strain and its safety as a brewing microorganism, it is attracting attention as a host for heterologous protein production (Biotechnology, 6, 1419 (1988), Japanese Patent Application Laid-Open No. Sho 62-27).
2988). From our research, A.I. In the production of a heterologous protein using oryzae, it was confirmed that if a gene of a near species such as the genus Aspergillus was used, the productivity would be further increased. In particular, A. oryzae gene,
When expressed under the control of a high expression promoter of oryzae, it was found that a very large amount of protein was produced (Japanese Patent Application No. 11-154271, Japanese Patent Application No. 2000-36754). The present invention has succeeded for the first time in large scale production of metallothionein by using this system to efficiently express the metallothionein gene in Aspergillus oryzae.

The metallothionein gene isolated as described above can be used alone or as a tyrosinase gene of Aspergillus oryzae (melO gene: Biochem. Biophys. Acta., 1261).
(1), p.151, 1995) and the promoter of a glucoamylase gene (glaB) derived from Aspergillus oryzae which is specifically expressed in large amounts in solid culture (Japanese Patent Application No. 11-154271, Japanese Patent Application Laid-Open No. 11-243965). Using a high expression promoter, Oryzae, or as a fusion protein with other proteins such as Aspergillus glucoamylase under the control of such a high expression promoter, expressed in A. oryzae and secreted and produced outside the cells Thus, a high-purity target protein is produced in large quantities.

As a method for gene transfer, an ordinary method is appropriately used. For example, as a host, a niaD mutant (a mutant of Aspergillus oryzae unable to assimilate nitrate: a Nitrate Reductase-deficient strain, such as Aspergillus oryzae 1013-niaD (FERM)
P-17707)), a metallothionein gene as a target gene and a niaD gene as a transformation marker gene (Unkle, ES et al.,
Mol. Gen. Genet., 218, p.99-104, 1989). By excluding a heterologous gene such as a vector sequence during this gene transfer, a transformant of a self-cloning strain containing no heterologous gene can be obtained. By culturing the transformant, the target protein can be secreted in large amounts into or out of the cells.

[0015] Thus, according to the present invention, the metallothionein gene is transformed into A. oryzae, and the target protein was isolated from A. oryzae. Oryzae succeeded in mass production. Furthermore, when the heterologous gene is introduced, Different type D other than oryzae
By adopting a method that does not contaminate NA, a self-cloning strain is obtained, and a remarkable effect is obtained that the strain is excluded from the regulation of recombinant microorganisms. Therefore, the metallothionein produced by the present invention can be used not only for industrial applications but also for foods and drinks, pharmaceuticals, cosmetics, and the like.
Hereinafter, the present invention will be described more specifically.

A clone showing homology to the metallothionein gene of Neurospora crassa was extracted from a database in which the nucleotide sequence of the cDNA library was comprehensively determined. Using this cDNA clone as a probe, oryzae lambda EMB
Positive clones containing the chromosomal metallothionein gene were selected from the L3 phage library. Using the obtained positive clone as a template, the entire nucleotide sequence of 323 bp including the metallothionein coding region was determined. As a result, the metallothionein gene contains one intron, the amino acid sequence of the protein deduced from the gene is 29 residues,
Neurospora crassa homology with metallothionein was 66% at the amino acid level.

The entire nucleotide sequence of the gene determined as described above is shown in SEQ ID NO: 2 (FIG. 2), and the amino acid sequence of the protein deduced from this nucleotide sequence is shown in SEQ ID NO: 1 (FIG. 1).

Next, A. The entire coding region of the glucoamylase gene glaB of Aspergillus oryzae is ligated downstream of the melO promoter, which is a high expression promoter in oryzae, and immediately thereafter, the coding region of this gene is ligated. did. As shown in FIG. o
The entire coding region of the glucoamylase gene glaB of Aspergillus oryzae and the coding region of the metallothionein gene were ligated downstream of the melO promoter to the Ryzae gene expression vector, and introduced into an A. oryzae niaD mutant. As a result, under the control of the melO promoter, about 2 g / 1 L-broth of the metallothionein glucoamylase fusion protein was secreted into the medium.
This enzyme protein has a purity of 99% and does not require purification. In addition, the metallothionein glucoamylase fusion protein was examined for its ability to adsorb heavy metals, and as a result, the activity of adsorbing zinc and cadmium was confirmed.

Among the obtained transformants, one of the suitable transformants was sent to the National Institute of Advanced Industrial Science and Technology
Deposited as ERM P-18152. The nucleotide sequence of the melO promoter is shown in SEQ ID NO: 5 and FIG. 5 and is the glucoamylase gene B of Aspergillus oryzae.
The nucleotide sequence of (glaB) is shown in SEQ ID NO: 6 and FIG.

From the above results, it was confirmed that the cloned gene fragment encodes a protein having metallothionein activity. In addition, using this gene, it is possible to produce a metallothionein glucoamylase fusion protein extracellularly in a highly secreted manner unlike conventional techniques, and the produced protein is used in the field of bioremediation such as recovery of harmful heavy metals in the environment. It was also confirmed that it was possible to use it. Hereinafter, examples of the present invention will be described.

[0021]

Example 1 Cloning of metallothionein gene cDN prepared based on mRNA obtained from liquid culture
From the A library (EST library), Neuro
A clone SH670 having high homology to the metallothionein gene of ospora crassa was selected. Aspergillus oryzae O-1013 using lambda EMBL3 phage
Co., Ltd. (Deposited by the National Institute of Advanced Industrial Science and Technology
The metallothionein gene was cloned from the chromosome gene library of ERM P-16528) by plaque hybridization.

In order to prepare a probe based on the EST sequence, the following two kinds of oligonucleotide primers were synthesized, and PCR was performed using the genome of the oryzae RIB40 strain as a template, and the reaction product was fluorescently labeled by a random priming labeling method to obtain a probe. Of the two types of oligonucleotide primers, one is a sense primer and its base sequence is shown in SEQ ID NO: 3 (FIG. 3), and the other is an antisense primer and its base sequence is SEQ ID NO: 4
(FIG. 4).

According to this method, a clone λMT88 hybridizing with the above probe was isolated from about 10,000 phage clones. The above oligo DNA
Was used as a primer to determine the DNA base sequence by the dideoxy method, and it was confirmed that this clone was indeed the target metallothionein gene.

[0024]

Example 2 Determination of the Base Sequence of the Metallothionein Gene The entire base sequence of the metallothionein gene was determined using the positive clone λMT88 strain as a template. A 323 bp sequence was determined by combining the promoter portion, the coding region and the terminator portion (SEQ ID NO: 2: FIG. 2). Of the above sequence, the position from position 1 to position 63 is the promoter portion, and the position from position 64 to position 2 is
The region up to position 10 is the coding region and 21
The terminator portion extends from the position 1 to the position 323.

From the result of the homology search with Neurospora crassa, it was found that the metallothionein gene had one intron, its coding region was 147 bp, and the protein estimated from the nucleotide sequence was 29 residues. It became clear. (SEQ ID NO: 1: FIG. 1).

[0026]

Example 3 Introduction of Metallothionein Gene into Aspergillus oryzae Downstream of melO promoter which is a high expression promoter of Aspergillus oryzae (its salt sequence is shown in SEQ ID NO: 5 (FIG. 5))
Aspergillus glucoamylase gene glaB (Japanese Unexamined Patent Publication No.
243965: The nucleotide sequence of which is shown in SEQ ID NO: 6 (FIG. 6) (including the start codon ATG)
584 bp), and immediately thereafter, the coding region 147 bp of the metallothionein gene (metA) obtained above was ligated. Further, this fusion gene (a gene encoding a metallothionein glucoamylase fusion protein) was transformed into a koji mold expression vector pIN9.
No. 3 metallothionein glucoamylase fusion protein expression plasmid pBMETL inserted into the PstI site
1 was constructed (FIG. 7).

This plasmid pBMETL1 was transformed into A. o
ryzae niaD mutant strain Aspergillus oryzae 1013-
It was introduced into niaD (FERM P-17707 deposited by National Institute of Advanced Industrial Science and Technology).
The strain whose nitrate utilization ability was restored was selected as the transgenic strain. The resulting transgenic strain was subjected to liquid culture in a Tzapek-Docs medium, and as a result, the metallothionein glucoamylase fusion protein was extracellularly added at about 2 g / 1 L-.
Broth was produced (Table 1). The purity of the product is 9
9%.

(Table 1) Secreted metallothionein glucoamylase fusion protein of transformant in Czapek-Dox medium融合 Metallothionein glucoamylase fusion protein (g / 1L-broth) 対 照 Control strain ND transformation Body 2.1

Next, the heavy metal adsorption activity of the metallothionein glucoamylase fusion protein was measured (Table 2). As a result, this fusion protein showed cadmium and zinc adsorption activities. The measurement of the heavy metal adsorption activity was performed using an atomic absorption spectrophotometer, Aanalyst 800 manufactured by PerkinElmer.
And according to the method of use.

(Table 2) Heavy metal content of metallothionein glucoamylase fusion protein ────────────────────────────── heavy metal content ( Metal atom g / mol protein) ────────────────────────────── cadmium 0.8 zinc 1.1 ───── ──────────────────────────

As a result, it was confirmed that the transformant into which the metA gene had been introduced encodes a protein having a very high heavy metal-trapping activity outside the cells, which was then transferred to Aspergillus oryzae ME.
It was named L-META and deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-18152.

Although the technique for expressing and producing metallothionein as a metallothionein glucoamylase fusion protein has been described in detail above, only metallothionein can be obtained by separating metallothionein and glucoamylase according to a conventional method. In addition, when an expression vector in which the glucoamylase gene glaB is deleted from the fusion protein expression vector is introduced into a host, only metallothionein can be expressed and produced alone. Furthermore, by using a gene other than glaB, the metallothionein fusion protein can be easily secreted and produced outside the cells.

[0033]

According to the present invention, it has become possible for the first time to secrete and produce a large amount of metallothionein inside or outside the cells. And, in particular, according to the present invention, by expressing metallothionein as a fusion protein with another protein, extracellular secretion becomes possible for the first time, and furthermore, this fusion protein has an excellent heavy metal capturing activity like metallothionein alone. It has been confirmed that industrial production of metallothionein is possible for the first time.

Since the metallothionein fusion protein has the ability to adsorb and bind to heavy metals similarly to metallothionein, harmful heavy metals in the environment can be captured and removed, and can be useful or obtained. The desired heavy metal can be captured, concentrated, separated and extracted. In the present invention, as the heavy metal,
Cadmium, zinc, copper, mercury, silver, iron, manganese, chromium, lead and other heavy metals are all included.

Therefore, according to the present invention, it becomes possible for the first time to produce and secrete metallothioneine of Aspergillus in large quantities, and it can be widely used in the field of bioremediation, which has been difficult to utilize conventionally. Also, since the present invention produces koji mold enzymes with koji mold, the enzyme protein produced is extremely safe and can be applied not only to the bioremediation field, but also to the food, pharmaceutical, and cosmetic industries. Technology.

[0036]

[Sequence List] SEQUENCE LISTING <110> Gekkeikan Inc., Ltd. <120> Metallothioneine-encoding Gene <130> 6372 <141> 2001-2-28 <160> 6 <210> 1 <211> 29 <212> PET <213> Aspergillus oryzae <400> 1 Met Gly Cys Asp Cys Gly Asn Thr Cys Gly Cys Ser Gly Pro Gln Ser 5 10 15 Cys Thr Cys Gly Gln Ser Cys Gln Cys Lys Asn Cys Gly 20 25 29 <210> 2 <211 > 323 <212> DNA <213> Aspergillus oryzae <400> 2 tctctatcta cttagtacta catcttacat attcccatct acaacattac ctgatacttc 60 acgatgggct gtgattgcgg aaatacctgc ggttgctccg gtccccagag ctgcacttgt 120 ggccagtcct gccagtgcaa gaactgcgga gtaagtatag ctatggaata gttgatttcc 180 atataacgct gattatttaa aaagaagtga tccattcgtc cgcacttgat tacatacgag 240 gaatccgatg aagtttacga tgttgaatga aggggatgga aatgatttgg agtattgtct 300 gggcgtgatt ttggacttta ttg 323 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <400> 3 ccgtttaaag agagtgtatt aagtagtatc 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <400> 4 ctctatctac ttagtactac atcttac 27 <210> 5 <211> 1173 <212> DNA <213> Aspergill us oryzae <400> 5 gcttgccttg gctcaaatcg ttcatgacac ccatctaggc catggcgcct gtagagcagg 60 ttacatttca tggccggtta atccgaatcc agtgcttgca catgtagcgc cacatggtct 120 gtgctattct attctgtgtt ataatagtgt gatttattgc gtttgggcgt ttcagttgat 180 tcgactggcc ttgcacatta ctctcgcatt ccacagctgg ctggaggagt tatctttact 240 tcttctttgt gactgtggct gcatgaggcg cttagtatac tatcagctga tactatgttg 300 aaactgaatc acggtgcttg aaggtctgcg tgaagtggtt cattgggctg tgatattaac 360 cgcagcctgt ctagaactat gactagacgg agcgccaaga atggacgaca acaggaatac 420 tgcccagcta gccacagctg aatcctaaag aagtttgcca gccctcgtat tcctatcctg 480 catggacggc aacattgccc tgacgagcta aattaggccg cagcgctagt attagaatga 540 actacggtag caatgagggg aacgcccaca agccaattaa cagcgctagt cttgatatga 600 cgggcctagc cttaattacg gggtactgtg aggacgttgt gcctgctgca attgtctatc 660 cgtgccgacg gtgttgacag ccactagcca ttcagctcgc cacactttca accccacacc 720 tcaaagtaag acctaaactt attttggact tccttgcagc tactatgctg tcactgttat 780 ttgactggac atgacatgca gtatcatggc gccaataaag agagtatctc gagagtttca 840 ttg catcgta ggaaaggctt gcattccggt gttgccggga aagggatcat tggtaatgcg 900 tagttgtttt gtctagctgt gatgccgggc tttgatggac ggaggacctg gagtgcagct 960 cttcatgcaa agcccgagat agactgattt gtaacatgtg tgatgcgtat cattcattat 1020 caatacgtct cgtggatatt taagaagggc gacagtcgtg tgaatatccg ctacttcaag 1080 ttcaaaacat cattcctacg aaaaggaaaa ccacagcttc cgcttcaaag ccctagtcaa 1140 cactagttca tcttctgatt actttggttc aca 1173 <210> 6 <211> 3000 <212 > DNA <213> Aspergillus oryzae <400> 6 gtagtattct gcactggtat ggaaccctcc caagccaggc tagctaaatt cactcctaga 60 ctcattctta gagtagattc aaccagtcgt acttgcaagg gtttcctgtg gaaaagaaca 120 accagtcgaa gtggtggagg tcatgtgatg acgtcgaaga gatggatgag tcataacgtc 180 atgaactttg ctggtttctt caacttctcc acatctcact tncaggtacc cgaggataat 240 ccaaggagct ctatggctat agctgtacga gtgctacggc atgccgtatt caactcaagc 300 taacctaacc aaatacaaaa cggcagcaca tgccgatgca taatgggagc tcaagacatt 360 atcgtgtccg gccaaatggc ggcgaatcgg ccattcaccg aacttggggc atctatctca 420 aacgatgaga cctttacttg gcataggaaa gattgttaat gggtgaagca tg ctcctagt 480 tgtatggccg atgtgcttcc ccgatcacaa ccgaaggttt gtacatcact ccactccgcc 540 ccgatcctgg cttaacctaa acacagtcaa gaataatagt ccgccttcta agagcattaa 600 gactcgacta ggactcagag gttcagtccg atggggtttc caccagtgag aactaagaga 660 atggcggcac gggcagatgt cgggatgatc acttttagtc gctccgacgc aatatcgatt 720 tcaattggat ccgccacgct gcatcccgga aattaatacc ggatctgctt catgatgggg 780 cctttggctg cggaaaactg ctgcgagtct cagtaatctc cgactgatcg gcccgctgcc 840 tgttggttaa tgtcatgcag cccgttgcgg atcggttcac tgaattatct acgttacgtc 900 taggtcgcgc tataaagaga cctgaaattc tcacttcgaa tgaggcagcc aacaaacagc 960 tacagtcatt tcttgtttct tggaagtcac caccatcatg cggaacaacc ttcttttttc 1020 cctcaatgcc attgctggcg ctgtcgcgca tccgtccttc cctatccata agaggcagtc 1080 ggatctcaac gccttcattg aggcacagac acccatcgcc aaacagggcg tcctcaataa 1140 tatcggcgct gatggcaagc ttgttgaggg ggctgccgct ggtatcgttg tagcctcccc 1200 atccaagagt aatcccgact gttcgtacaa tcctaccctc aagaccgcat gatattacca 1260 cagagctaac tatatataga cttctacacc tggacgcgcg acgctggcct caccatggaa 1320 g aagtgatag agcaattcat cgggggagat gcgactctcg agtccacaat ccagaattat 1380 gttgactctc aagcgaacga gcaggcagtc tccaacccat caggcggcct gtcggatggc 1440 tcgggtcttg ctgaacccaa attttacgtc aatatctctc aattcaccga ttcttggggc 1500 cgaccccagc gcgacgggcc agccttacgt gcttccgctt tgatcgcata tggcaactct 1560 ctgatttcca gcgacaaaca atctgttgtc aaagctaaca tctggccaat tgtccagaat 1620 gacttgtctt atgtgggtca atactggaac cagaccgggt ttgatctttg ggaagaggtt 1680 cagggcagct ccttcttcac tgttgctgtg cagcacaaag ccttggtgga gggcgatgcg 1740 tttgcaaagg cactcggaga ggaatgccag gcatgctccg tggcgcctca aatcctctgc 1800 catcttcagg acttctggaa tgggtctgct gttctttcta acttaccaac caatgggcgc 1860 agtggactgg ataccaactc tcttttgggc tccattcaca cttttgatcc agccgccgct 1920 tgtgatgata caacattcca gccctgctcc tctcgcgccc tgtcgaacca taagcttgtg 1980 gttgactctt tccggtcggt ctacggtatc aacaatggac gtggagcagg aaaggccgcg 2040 gcagtgggcc cgtacgcaga ggacacctat cagggaggca atccatggtt ggtactctgt 2100 ctcatatcca aagcttaaac taatgaatat taggtatctt accaccctgg tcgctgcgga 2160 attgctc tac gacgccttgt atcagtggga caaacaaggt caagtgaacg tcactgaaac 2220 ttcccttccc ttcttcaagg acctctccag caatgtcacc accggatcct acgccaagtc 2280 ttcctcagcc tatgagtcgc ttacgagcgc tgtcaagacc tacgcagacg gcttcatctc 2340 cgttgtccag gagtatactc ccgatggcgg tgctttggct gagcagtaca gtcgggacca 2400 gggcacccca gtttcggcat ccgatctgac ttggtcttat gcagctttct tgagtgctgt 2460 tggacgacga aacggcactg tccctgctag ctggggctct tccacggcca acgcagttcc 2520 aagccaatgt tcggggggta cagtttctgg aagttacact accccaactg ttgggtcgtg 2580 gtagatgtac tttccagtgc gtgtagtcta ctctgacctc gtgtcacgat tgttgctttt 2640 gcctgtctaa atgcgaccgt gctgtgcatg tttgttaaat actgtcattc atctttgttt 2700 caacaacaaa gattacatca attagtgcta gctagacaat aacttttaca gttgcaacgt 2760 tagtcctagt attatacatc tcaccggatc ctcttcaaac ttcacggggt aaccaaaaga 2820 aagtaacaag actaagccta ttgatactgt ggttctaatc ttattttagt ttcctgtacg 2880 tccactgcaa tcaaactaag tatacatact acatcctagt atcctacacc gtatccttca 2940 accctagaac ctcccgctag gtatgtactg tagatcgatt atccggagat tccccgccac 3000

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of metallothionein.

FIG. 2 shows the base sequence of a metallothionein gene.

FIG. 3 shows sense primers.

FIG. 4 shows antisense primers.

FIG. 5 shows the nucleotide sequence of the melO promoter.

FIG. 6 shows the nucleotide sequence of the Aspergillus glucoamylase gene (glaB).

FIG. 7 shows the construction of a metallothionein glucoamylase fusion protein expression plasmid pBMETL1 and its restriction enzyme map.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) // B01J 20/24 C12N 9/34 4H045 C12N 9/34 (C12N 1/15 (C12N 1/15 C12R 1:69) (C12R 1:69) C12N 15/00 ZNAA (72) Inventor Yoji Hata 300 Kataharacho, Fushimi-ku, Kyoto, Japan Tsukikeikan Co., Ltd. (72) Inventor Shoji Kawado 24 Tsukiba-Koyanagicho, Fushimi-ku, Kyoto City Tsukikeikan Co., Ltd. In the laboratory (72) Kuniyasu Goto Inventor Kuniyasu Goto 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Pref. National Tax Agency Brewing Research Laboratory F term (reference) 4B024 AA17 BA12 BA80 CA04 DA11 EA04 GA11 GA19 HA01 HA12 4B050 CC05 DD03 LL10 4B064 AG01 CA05 CA19 CC24 DA16 4B065 AA63X AA63Y AB01 AC14 AC15 BA02 BA24 CA24 CA32 CA54 4G066 AB29A AB29B AC03B CA46 CA47 DA07 4H045 AA10 AA20 AA30 BA10 BA41 CA15 DA89 EA61 FA74

Claims (9)

[Claims] 1. A protein having an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing and having a metallothionein-like heavy metal-capturing activity. 2. A DN comprising a gene encoding the protein of claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2.
A. 3. A recombinant vector comprising at least the coding region of the DNA according to claim 2. 4. The recombinant vector pBMETL1. 5. A transformant obtained by introducing the recombinant vector according to claim 3 or 4 into Aspergillus. 6. An Aspergillus oryzae MEL-META (FER)
MP-18152). 7. A method for producing a protein having a metallothionein-like heavy metal-capturing activity, which comprises using the transformant according to claim 5 or 6. 8. The method according to claim 7, wherein a protein having a metallothionein-like heavy metal-capturing activity is secreted and produced outside the cells as a fusion protein. 9. A method for capturing heavy metals, comprising using the protein according to claim 1.