JP2003250562A - HEAT-RESISTANT alpha-GALACTOSIDASE GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new thermally stabler α-galactosidase protein derived from a strictly anaerobic thermophilic bacterium, and to provide a gene encoding the protein, a recombinant vector containing the gene, a transformant containing the recombinant vector, and to provide a method for producing the protein by using the transformant. <P>SOLUTION: The α-galactosidase is produced by culturing an α-galactosidase derived from Clostridium stercorarium of the strictly anaerobic thermophilic bacterium, or by culturing the transformant transformed by the vector containing the gene (aga36A gene) encoding the above activities. The sugar-transferring reaction can be carried out at high temperatures because the enzyme is stable against the heat. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel α-galactosidase protein derived from absolutely anaerobic thermophile, a gene encoding the protein, a recombinant vector containing the gene, and a transformation containing the recombinant vector. And a method for producing the protein using the transformant.

[0002]

BACKGROUND OF THE INVENTION α-Galactosidase is a bacterium, a mold,
It is an enzyme that exists widely in yeasts, plants, and animals and catalyzes the reaction of hydrolyzing α-D-galactoside bonds to release D-galactose. This enzyme has been isolated and studied from various organisms since it was extracted from yeast as an enzyme (melibiase) that hydrolyzes the reducing disaccharide melibiose. Then, α-galactosidase is used for decomposing and removing raffinose in a sugar solution that inhibits the crystallizing action of sucrose in the sugar manufacturing process in the sugar beet sugar manufacturing industry.

[0003] In particular, α-galactosidase derived from guar, purple horse mackerel, coffee and fenugreek plants
[Japanese Patent Publication 4-34398, Japanese Patent Publication 8-22396], Penicillium (Penic
illium) mold-derived α-galactosidase [Japanese Patent Publication 7-36752], Mortierella (Mortierella) mold-derived α-galactosidase [JP-A-61-274695],
Α-Galactosidase derived from a bacterium belonging to the genus Thermotoga (Biotec. Bioeng., 52, 332, 19)
96], α-galactosidase derived from mold belonging to the genus Aspergillus [Biochem. Soc. Trans. 19, 269]
S, 1991; EP-A-0121960], and α-galactosidase derived from mold belonging to the genus Monascus [Appl. Enviro]
n. Microbiol. 52, 1147, 1986] has been shown to directly act on guar gum galactomannan and exhibit galactose-releasing activity, and its use in industrial applications such as food processing and guar gum modification. Is expected.

Regarding the gene encoding α-galactosidase, Escherichia coli [Nucleic Acid Res. 23, 210]
5,1995], yeast [Yeast 10: 1559, 1994], Aspergillus niger [Mol. Gen. Genet. 233,404, 1992; Tokumeihei 8-50864
4], Streptococcus mutans
mutans) [J. Gen. Microbiol. 137, 757, 1991], guar seed [Plant Mol. Biol. 13, 541, 1989; Japanese Patent Publication 8-2.
2396], coffee Arabica [Gene 140, 227, 1994], murine [Gene 166, 277, 1995], and human [Nucleic Acids Re
s. 17, 3301, 1989] has already been cloned and the nucleotide sequence has been determined.

Α-Galactosidase is aerobic (facultative)
In addition to fungi, plants and animals,
The absolutely anaerobic bacterium Clostridium joseui (Clostri
(Japanese Patent Laid-Open No. 2000-245)
479].

[0006]

However, in the sugar conversion reaction by α-galactosidase, the reaction solution is often accompanied by high viscosity, which may be an obstacle to industrial practice. In this case, the viscosity is usually lowered at a higher temperature, but ordinary α-galactosidase is not sufficiently stable against heat. The present invention provides a novel heat-stable α-galactosidase protein derived from absolutely anaerobic thermophile, a gene encoding the protein, a recombinant vector containing the gene, and a transformant containing the recombinant vector. An object of the present invention is to provide a method for producing the above protein using the transformant.

[0007]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have found that the obligately anaerobic thermophilic bacterium Clostridium stercoralium (Clostridium).
stercorarium) α-galactosidase gene (aga36A)
The present invention has been completed and the present invention has been completed.

That is, the present invention is the following recombinant protein (a) or (b). (a) A protein containing the amino acid sequence represented by SEQ ID NO: 2. (b) A protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which at least one amino acid has been deleted, substituted or added, and having α-galactosidase activity.

Furthermore, the present invention is a gene encoding the following recombinant protein (a) or (b). (a) A protein containing the amino acid sequence represented by SEQ ID NO: 2. (b) A protein comprising an amino acid sequence represented by SEQ ID NO: 2 in which at least one amino acid has been deleted, substituted or added, and having α-galactosidase activity.

Furthermore, the present invention provides the following (c) or (d) DN:
It is a gene containing A. (c) A DNA containing the nucleotide sequence represented by SEQ ID NO: 1. (d) DNA that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having α-galactosidase activity
(d) DN that hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and encodes a protein having α-galactosidase activity
A.

Further, the present invention is a recombinant vector containing the above gene. Furthermore, the present invention is a transformant containing the above recombinant vector. Furthermore, the present invention is a method for producing the above protein, which comprises culturing the above transformant in a medium and collecting a protein having an α-galactosidase activity from the obtained culture. Furthermore, the present invention is a method for producing a protein, which comprises culturing Clostridium stercoralium in a medium and collecting a protein having an α-galactosidase activity from the obtained culture.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. The present inventors have found α-galactosidase activity in the culture supernatant of the obligately anaerobic thermophilic bacterium Clostridium stercoralium. Then, when shotgun cloning was performed, a clone having α-galactosidase activity was found, and as a result of gene analysis, family 3
6: α-galactosidase gene belonging to clan GH-D (aga
36A gene). This aga36A gene can be cloned as follows.

1. Cloning of aga36A gene (1) Preparation of genomic DNA library The genomic DNA of Clostridium stercoralium is G
It can be prepared from anaerobically cultivated Clostridium stercoralium using S medium, PY medium, VL medium and the like by a commonly used method. For example, bacterial cells obtained by liquid culture under anaerobic conditions are recovered by centrifugation, suspended in an appropriate buffer solution (for example, TE buffer), and then chemically suspended by a surfactant (for example, SDS, CTAB). Cells are destroyed and nucleic acids are extracted by the treatment method. Then, the genomic DNA can be prepared by degrading RNA by ribonuclease treatment and then precipitating with alcohol.

Next, the obtained genomic DNA is digested with an appropriate restriction enzyme (for example, Sau3AI etc.), treated with chloroform, and the DNA fragment is recovered by ethanol precipitation. The obtained DNA fragment is fractionated by agarose gel electrophoresis or the like, and then ligated to an appropriate vector (eg, phage vector such as λgt10, λgt11, plasmid vector such as pBR322, pUC19, pBluescript, cosmid vector such as Lawrist4). Finally, the ligation was transformed into E. coli (e.g. DH5α
Strain, XL1blue strain, C600 strain, JM109 strain, etc.) to prepare a genomic DNA library. In addition, Clostridium stercoralium used in the present invention was stored in the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1-3, Higashi 1-3, Tsukuba City, Ibaraki Prefecture)
ridium stercorarium F-9 deposited in FERM P-18745.

(2) Cloning of aga36A gene of the present invention The aga36A gene of the present invention can be screened from the transformants obtained in the above (1) using the α-galactosidase activity as an index. That is, the book α
-Since galactosidase has heat resistance, α-galactosidase is detected after the host α-galactosidase is completely inactivated by heat treatment. For example, when the host is Escherichia coli, heat treatment at 60 ° C. for about 10 to 20 minutes is sufficient. After that, overlaying soft agar containing 4-methylumbelliferyl-α-D-galactopyranoside and leaving it at about 60 ° C, the fluorescence of 4-methylumbelliferone under ultraviolet irradiation causes α-galactosidase. A clone having activity can be selected. In addition, disrupted cultures of these clones were treated with pNP-Gal (p-nitrophenyl-α-D-galactopyranoside), disaccharide α-D-melibiose, trisaccharide D-raffinose, and galactomannan. A clone carrying aga36A can be selected by investigating whether it acts on a certain guar gum or the like.

Then, an insert DNA fragment is prepared from the obtained positive strain and the nucleotide sequence is determined according to a conventional method. The determination of the nucleotide sequence can be performed by known methods such as Maxam-Gilbert's chemical modification method, or dideoxynucleotide chain termination method using M13 phage, but usually an automatic nucleotide sequencer (for example, model 4000L manufactured by LI-COL,
Sequencing is performed using ALFII type (Pharmacia). And the determined nucleotide sequence is Genetyx, DNASIS
The open reading frame is searched using a DNA analysis software such as, and the obtained deduced amino acid sequence is searched for in a database to check the homology with known proteins.

The nucleotide sequence of the aga36A gene of the present invention is shown in SEQ ID NO: 1 and the amino acid sequence of the aga36A protein of the present invention is shown in SEQ ID NO: 2 as long as the protein consisting of this amino acid sequence has α-galactosidase activity. Mutations such as deletion, substitution, and addition may occur in at least one amino acid in the amino acid sequence. For example, at least one, preferably about 1 to 20, more preferably 1 to 10 amino acids of the amino acid sequence represented by SEQ ID NO: 2 may be deleted, or the amino acid represented by SEQ ID NO: 2 At least 1, preferably about 1 to 20, more preferably 1 to 10 amino acids may be added to the sequence, or at least one of the amino acid sequences represented by SEQ ID NO: 2, preferably 1 to About 20 amino acids, more preferably 1 to 10 amino acids may be substituted with other amino acids.

Further, a DNA which can hybridize with the above gene under stringent conditions is also included in the gene of the present invention. Stringent conditions include, for example, sodium concentration of 15 to 150 mM, preferably 15 to 30 mM.
And the temperature is 37 to 80 ° C., preferably 50 to 65 ° C. In addition, to introduce mutations into genes, Kunkel
Method, a known method such as Gapped duplex method or a method similar thereto, for example, a mutagenesis kit using a site-directed mutagenesis method (for example, Mutant-K (TAKARA), Mu
tant-G (manufactured by TAKARA), etc., or TAKARA
It can be performed using LA PCR in vitro Mutagenesis series kit of the same company.

Once the nucleotide sequence of the gene of the present invention has been determined, thereafter, it is chemically synthesized or the cD of the gene of the present invention is determined.
The gene of the present invention can be obtained by PCR using NA or genomic DNA as a template, or by hybridizing with a DNA fragment having the base sequence as a probe.

2. Preparation of Recombinant Vector and Transformant (1) Preparation of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating (inserting) the gene of the present invention into an appropriate vector.
The vector for inserting the gene of the present invention is not particularly limited as long as it can be replicated in a host, and examples thereof include plasmid DNA and phage DNA. Plasmid
As DNA, a plasmid derived from Escherichia coli (for example, pBR32
2, pBR325, pUC118, pUC119, pUC18, pUC19, pCBD-C, p
Bluescript, etc.), a plasmid derived from Bacillus subtilis (eg pUB1
10, pTP5, etc.), yeast-derived plasmids (eg YEp13, YE
p24, YCp50, YIp30 and the like), and the phage DNA includes λ phage and the like. Furthermore, animal viruses such as retrovirus and vaccinia virus, and insect virus vectors such as baculovirus and togavirus can also be used.

To insert the gene of the present invention into a vector, first, the purified DNA is cleaved with an appropriate restriction enzyme,
A method of inserting into an appropriate vector DNA at a restriction enzyme site or a multi-cloning site and ligating to a vector is adopted. The gene of the present invention needs to be incorporated into a vector so that the function of the gene is exerted. Therefore, in the vector of the present invention, in addition to the promoter, the gene of the present invention, optionally a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD sequence)
Those containing such can be linked. As the selection marker, for example, ampicillin resistance gene,
Neomycin resistance gene dihydrofolate reductase gene,
Etc.

(2) Preparation of Transformant The transformant of the present invention can be obtained by introducing the recombinant vector of the present invention into a host so that the target gene can be expressed. Here, as the host, the
There is no particular limitation as long as it can express DNA. For example, Escherichia coli and other genus Escherichia, Bacillus subtilis
Pseudomonas, Pseudomonas, etc.
Bacteria belonging to the genus Pseudomonas such as putida) and the genus Rhizobium such as Rhizobium meliloti (Saccharomyces cerevisiae).
es cerevisiae), Schizosaccharomyces pombe (Schizos
yeasts such as accharomyces pombe), COS cells, CH
Examples thereof include animal cells such as O cells, and insect cells such as Sf9 and Sf21. When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention is capable of autonomous replication in the bacterium and is composed of a promoter, a ribosome binding sequence, the gene of the present invention, and a transcription termination sequence. Is preferred. Moreover, the gene which controls a promoter may be contained. Examples of Escherichia coli include Escherichia coli K12 and DH1, and examples of Bacillus subtilis include Bacillus subtilis (B
acillus subtilis) MI 114, 207-21 and the like.

Any promoter may be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli or phage such as trp promoter, lac promoter, PL promoter and PR promoter are used. An artificially designed and modified promoter such as tac promoter may be used. As a method of introducing the recombinant vector into the bacterium,
The method is not particularly limited as long as it is a method of introducing DNA into bacteria. For example, a method using calcium ions [Cohe
n, SN et al .: Proc. Natl. Acad. Sci., USA, 69: 2
110 (1972)], electroporation method and the like.

When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe
e), Pichia pastoris and the like are used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast, for example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. Is mentioned.

The method for introducing the recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. For example, the electroporation method [Becker, DM et
al .: Methods. Enzymol., 194: 182 (1990)], spheroplast method [Hinnen, A. et al .: Proc. Natl. Acad.
Sci., USA, 75: 1929 (1978)], lithium acetate method [Itoh,
H .: J. Bacteriol., 153: 163 (1983)] and the like.

When an animal cell is used as a host, monkey cell CO
S-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, human GH3, human FL cells and the like are used. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter and the like are used, and human cytomegalovirus early gene promoter and the like may be used. Examples of the method for introducing the recombinant vector into animal cells include the electroporation method, calcium phosphate method, lipofection method and the like. When using insect cells as hosts, Sf9 cells, Sf2
One cell is used. Examples of the method for introducing the recombinant vector into insect cells include the calcium phosphate method, lipofection method, electroporation method, and the like.

3. Production of the Protein of the Present Invention The protein of the present invention has an amino acid sequence encoded by the α-galactosidase gene or the catalytic domain gene of α-galactosidase of the present invention, or at least one amino acid in the amino acid sequence is mutated as described above. Has an introduced amino acid sequence.
Furthermore, fusion proteins of these proteins and cellulose-binding proteins or β-galactosidase proteins are also included in the proteins of the present invention. The protein of the present invention can be produced from the culture of a bacterium belonging to the genus Clostridium or the transformant culture obtained in the above 2 as follows. Here, the "culture" means
It means any of the culture medium supernatant after culturing, the cells obtained by culturing, or the disrupted product of the cells. The method for culturing a bacterium belonging to the genus Clostridium (for example, Clostridium stercoralium) or the transformant obtained in the above 2 is performed according to a usual method used for culturing those cells.

The medium may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the cells, and can efficiently culture the cells. May be used. Here, as the carbon source,
Glucose, fructose, sucrose, dextrin, carbohydrates such as starch, organic acids such as acetic acid and propionic acid, alcohols such as glycerol, ethanol and propanol, animal oil, molasses and the like are used. Further, as the nitrogen source, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of inorganic acids or organic acids such as ammonium phosphate or other nitrogen-containing compounds, peptone, meat extract, yeast extract, malt extract, Corn steep liquor or the like is used. Furthermore, as inorganic salts, potassium, sodium, calcium, magnesium, manganese, cobalt,
Salts of cations such as zinc, iron and molybdenum and anions such as sulfuric acid, phosphoric acid, hydrochloric acid and nitric acid are used.

In the case of anaerobically culturing a bacterium belonging to the genus Clostridium, a substance having a reducing action such as cysteine is added to the medium, and inoculation of the bacterium is carried out by nitrogen purging of oxygen remaining in the space above the incubator. It is preferable to carry out after sufficient removal. The anaerobic bacteria are cultured in an anaerobic box, an anaerobic jar, a test tube with a butyl rubber stopper, and the like. Further, when culturing the transformant, antibiotics such as ampicillin and tetracycline may be added to the medium, if necessary. Then, the cells are cultured until the α-galactosidase activity of the cells reaches the maximum value.

Further, when culturing bacteria belonging to the genus Clostridium, galactomannans such as guar gum, galactosides such as lactose, melibiose and raffinose, or isopropylthio-β-D-galactoside are added, and α-galactosidase per cell is added. Since it may be possible to increase the activity, these substances are added as necessary. Further, in the culture of the transformant, when the transformant is transformed with an expression vector using an inducible promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-β-D-
When culturing a microorganism in which thiogalactopyranoside (IPTG) or the like has been transformed with an expression vector using the trp promoter, indole acrylic acid (IAA) or the like may be added to the medium.

As a medium for culturing a transformant obtained by using animal cells as a host, generally used RPMI16
40 medium, DMEM medium, or a medium obtained by adding fetal bovine serum or the like to these mediums is used. Culturing is usually carried out in the presence of 5% CO2 at 37 ° C for 1 to 30 days. If necessary, an antibiotic such as kanamycin or penicillin may be added to the medium during the culture.

After culturing, when the α-galactosidase protein of the present invention is produced in the cells or cells, the cells or cells are disrupted to extract the α-galactosidase protein. The cells can be disrupted by using ultrasonic waves, a French press, a homogenizer using glass beads, or the like, but can also be used in combination with lysozyme or a freeze-thaw method. When the α-galactosidase protein of the present invention is produced outside the cells or cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, common biochemical methods used for isolation and purification of proteins, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, hydrophobic chromatography,
The protein of the present invention can be isolated and purified from the culture by using affinity chromatography or the like alone or in appropriate combination.

4. Measurement of α-galactosidase activity The activity of α-galactosidase can be measured as follows. That is, the enzyme solution is allowed to act on a disaccharide such as α-D-melibiose or a trisaccharide such as D-raffinose, and the resulting α-D-galactoside is detected by thin layer chromatography or the like. Also, pNP-Ga
It can also be performed by reacting a synthetic substrate such as l (p-nitrophenyl-α-D-galactopyranoside) with an enzyme solution and quantifying the released para-nitrophenol using a spectrophotometer. .

5. Antibodies Against Proteins of the Present Invention In the present invention, antibodies against the proteins of the present invention can also be prepared. "Antibody" means the whole antibody molecule or a fragment thereof capable of binding to the peptide of the present invention which is an antigen.
(For example, Fab or F (ab ′) 2 fragment), and may be a polyclonal antibody or a monoclonal antibody. The antibody of the present invention can be produced by any of various methods. Methods for producing such antibodies are well known in the art [eg Sambrook, J et al., Molecular
Cloning, Cold Spring Harbor Laboratory Press (198
See 9)].

(1) Preparation of Polyclonal Antibody against Protein of the Present Invention α of the present invention prepared by genetic engineering as described above
-Using a galactosidase protein or a fragment thereof as an antigen, this is administered to mammals such as humans, mice, and rabbits. The dose of the antigen per animal is 1 to 5 mg when no adjuvant is used, and 50 to 100 μg when an adjuvant is used. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant and the like. Immunity is mainly intravenous, subcutaneous,
It is performed by injecting into the abdominal cavity or the like. In addition, the immunization interval is not particularly limited, and immunization is performed 2 to 5 times, preferably 3 to 4 times at intervals of several days to several weeks, preferably at intervals of 2 to 4 weeks. And 12-16 days after the last immunization day,
Preferably, enzyme immunoassay (EIA)
y), the antibody titer is measured by radioimmunoassay (RIA) or the like, and blood is collected on the day when the maximum antibody titer is exhibited to obtain an antiserum. When purification of the antibody from the antiserum is required, it should be purified by appropriately selecting known methods such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or by combining these methods. You can

(2) Preparation of Monoclonal Antibody Against Protein of the Present Invention (i) Collection of Antibody-Producing Cells Using the protein of the present invention genetically engineered as described above or a fragment thereof as an antigen, a mammal, for example, It is administered to rats, mice, rabbits, etc. Animal of antigen 1
The dose per animal is 1 when no adjuvant is used.
~ 5mg, 25 ~ 100μg when using adjuvant
Is. Examples of the adjuvant include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminum hydroxide adjuvant and the like. Immunization is mainly performed by injecting intravenously, subcutaneously, or intraperitoneally. Further, the immunization interval is not particularly limited, and may be at intervals of several days to several weeks, preferably 2 to 4 weeks, 2 to
Immunization is performed 5 times, preferably 3 to 4 times. Then, antibody-producing cells are collected 12 to 16 days, preferably 14 days after the final immunization date. Examples of antibody-producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, but spleen cells or local lymph node cells are preferred.

(Ii) Cell fusion To obtain a hybridoma, cell fusion between antibody-producing cells and myeloma cells is performed. As the myeloma cells to be fused with the antibody-producing cells, commonly available cell lines of animals such as mice can be used. The cell line used has drug selectivity and cannot survive in HAT selective medium (including hypoxanthine, aminopterin, and thymidine) in the unfused state, and can survive only in a state fused with antibody-producing cells. Those having are preferred. Examples of myeloma cells include P3X63-Ag.8.U1 (P3U1), Sp2 / O, N
Mouse myeloma cell lines such as SI are mentioned. Next, the myeloma cells and antibody-producing cells are fused. For cell fusion, antibody-producing cells and myeloma cells are mixed at a ratio of 4: 1 to 1: 4 in a medium for animal cell culture such as serum-free DMEM or RPMI-1640 medium, and a cell fusion promoter is present. The fusion reaction is performed under. As the cell fusion promoter, polyethylene glycol having an average molecular weight of 1,500 daltons can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device that utilizes electrical stimulation (eg, electroporation).

(Iii) Selection and cloning of hybridoma The target hybridoma is selected from the cells after the cell fusion treatment. As the method, after appropriately diluting the cell suspension with RPMI-1640 medium containing fetal bovine serum, etc., spread about 0.8 cells / well on a microtiter plate, add the selective medium to each well, and then appropriately select the selective medium. Replace and culture. As a result, cells that grow from about 14 days after the start of culture in the selective medium can be obtained as hybridomas. Next, it is screened whether or not the target antibody is present in the culture supernatant of the grown hybridoma. The screening of hybridomas may be performed according to a usual method and is not particularly limited. For example, a part of the culture supernatant contained in the wells grown as hybridomas can be collected and subjected to enzyme immunoassay, radioimmunoassay or the like. Cloning of fused cells
This is carried out by the limiting dilution method or the like to finally establish a hybridoma that is a monoclonal antibody-producing cell.

(Iv) Collection of Monoclonal Antibody As a method for collecting a monoclonal antibody from the established hybridoma, an ordinary cell culture method, ascites formation method or the like can be adopted. In the cell culture method, the hybridoma is subjected to normal culture conditions (eg, 37 ° C., 5% CO 2 concentration) in an animal cell culture medium such as RPMI-1640 medium containing 10% fetal bovine serum, MEM medium or serum-free medium at 2%. Cultivate for ~ 10 days, and obtain the antibody from the culture supernatant. In the case of the ascites formation method, about 1 × 10 7 hybridomas are intraperitoneally administered to a mammal derived from a myeloma cell and an animal of the same species, and the hybridomas are proliferated in a large amount. Then, after 1 to 2 weeks, ascites or serum is collected. In the above-mentioned antibody collection method, when purification of the antibody is required, a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, or affinity chromatography is appropriately selected, or a combination thereof is used. Can be purified by.

After the polyclonal antibody or the monoclonal antibody is obtained in this manner, an affinity chromatography column is prepared by binding the polyclonal antibody or the monoclonal antibody to a solid carrier using the ligand as a ligand, and using the column, the above-mentioned collection source or The α-galactosidase proteins of the invention can be purified from other sources. Furthermore, these antibodies can also be used in Western blotting to detect the α-galactosidase protein of the present invention.

[0041]

[Example 1] Cloning of α-galactosidase gene (1) Preparation of chromosomal DNA According to a conventional method, Clostridium stercoralium (C
A chromosomal DNA of lostridium stercorarium) was prepared. That is, Clostridium stercorarium FERM P-18745 strain, 100ml of GS medium (0.45% yeast extract, 0.5% cellobiose, 0.1% cysteine hydrochloride, 2.09% MOPS, 0.15% KH2
PO4, 0.29% K2HPO4, 0.13% (NH4) 2SO4, 0.2% MgCl2, 0.
After anaerobically culturing in 03% CaCl2, 0.00025% FeSO4 · 7H2O, pH 7.4) at 60 ° C for 1 day, the cells were recovered by centrifugation. The obtained bacterial cells were mixed with 10 ml of TE buffer (10 mM Tris-HCl).
(pH 7.4), 1mM EDTA), and then add 1 ml of 10% SDS and
0.1 ml of 20 mg / ml proteinase K was added, and the mixture was incubated at 37 ° C for 1 hour. Then add 2 ml of 5M NaCl and stir well, then add 1 ml.
10% CTAB (cetyltrimethylammonium bromide)
-0.7M NaCl was added, and the mixture was kept at 65 ° C for 10 minutes. Then, the same amount of chloroform (containing 4% isoamyl alcohol) was added, and the mixture was stirred well and then centrifuged. Transfer the obtained supernatant to a sterilized centrifuge tube, add 2 volumes of ethanol, and bring it to room temperature for 1 hour.
It was left for 0 minutes. Then, the white precipitate was collected by centrifugation and dried in a desiccator under reduced pressure. Then
The cells were suspended in 1 ml of TE buffer, a ribonuclease A solution was added to 10 mg / ml, and the mixture was incubated at 37 ° C for 30 minutes. 0.1 ml
After adding 3 M sodium acetate (pH 8.0) and 2.2 ml of ethanol and mixing them well, the supernatant was removed by centrifugation.
The precipitate was dried in a desiccator and then dissolved in 0.5 ml of TE buffer to obtain a chromosomal DNA.

(2) Preparation of DNA library Using the genomic DNA obtained in the above (1), a chromosomal DNA library of Clostridium stercoralium was prepared. That is, the chromosomal DNA is restricted to the restriction enzyme Sau3AI.
After partially cutting with agarose gel electrophoresis with 0.4% agarose HI4 (Takara Shuzo), 5-10 kbp
Cut out the DNA fragment, Gene Clean II kit (Bio101)
Recovered by. Furthermore, in the presence of dG and dA, a 5'-overhanging end was partially repaired and synthesized with Klenow enzyme to prepare a chromosomal DNA fragment having a 2-nucleotide overhanging end. on the other hand,
The plasmid pBluescriptIIKS + (manufactured by Stratagene) was digested with a restriction enzyme XhoI and then recovered with a Gene CleanII kit to partially repair and synthesize a 5'-protruding end to obtain a plasmid having a 2-nucleotide protruding end. The above prepared chromosome
The DNA fragment and the above plasmid were ligated with T4 DNA ligase and introduced into Escherichia coli DH5α. At that time, the competent cells and the transformants were prepared by a conventional method using calcium chloride.

These transformants were treated with ampicillin (50
μl / ml), IPTG (isopropylthio-β-D-galactopyranoside; 40 μM), X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; 40 μg / ml ) Containing LB agar plate (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%
%, Agar 1.5%, pH 7.0; φ9 × 1.5 cm, round petri dish) and cultured at 37 ° C. Among the formed colonies, about 5,000 white colonies were collected.

(3) Selection of Clones Having α-Galactosidase Activity The LB agar plates were inoculated with the collected colonies, incubated overnight at 37 ° C. to form colonies again, and then treated at 60 ° C. for 15 minutes, The E. coli α-galactosidase was completely inactivated. Further, 0.5% agar containing 10 mM 4-methylumbelliferyl-α-D-galactopyranoside was overlaid and kept warm for 2 hours. Thereafter, a colony that fluoresces 4-methylumbelliferone under UV irradiation was used as a positive clone.

This positive clone was treated with 5 mL of LB medium (polypeptone 1.0%, yeast extract 0.5%, NaCl 0.5%, pH.
7.0) and incubating with shaking at 37 ° C. overnight. The bacterial cells collected by centrifugation were suspended in 5 mL of McIlvain's buffer pH7, sonicated for 2 × 30 seconds to disrupt the bacterial cells, and the disrupted bacterial cells were removed by centrifugation to obtain a clear solution. Obtained. Further, after deactivating the enzyme of Escherichia coli by heat treatment at 60 ° C. for 15 minutes, the insoluble matter was removed by centrifugation to obtain a crude enzyme solution. The α-galactosidase activity was confirmed using pNP-Gal (p-nitrophenyl-α-D-galactopyranoside) as a substrate. That is, 0.2 ml of McIlvain's buffer pH 7.0, 0.1 ml
10 mM pNP-Gal and 0.1 ml of the crude enzyme were mixed and reacted at 60 ° C., PNP release was measured by absorbance at 410 nm, and two strains having α-galactose activity were obtained.

Further, the effects of the disaccharide α-D-melibiose, the trisaccharide D-raffinose and galactomannan guar gum were examined. The hydrolyzate of these substrates was confirmed by using silica gel thin layer chromatography (SILHER G-25 manufactured by MACHERY-NAGEL) that was heat-treated at 110 ° C for 30 minutes, and the enzyme reaction was 80 μl of 1% (wt / vol). 40 μl of each substrate solution
The above crude enzyme solution and 80 μl of McIlvain's buffer pH 5.5 were mixed and reacted at 35 ° C. for 24 hours. The developing solvent for thin-layer chromatography is 1-propanol: nitromethane: water (5:
2: 3, vol / vol) was used. After the development, it was air-dried, and the color was developed by spraying sulfuric acid and treatment at 140 ° C for 5 minutes. One of the two strains released α-D-galactose from α-D-melibiose, D-raffinose, and guar gum, and the other strain released α-D-galactose from two substrates except guar gum. . From this, the former transformant that acts on guar gum was selected and subjected to the following steps.

(4) Preparation of recombinant plasmid The transformant selected in (3) above was treated with ampicillin (50
μl / ml) in 100 ml of LB medium was cultured at 37 ° C. overnight and the cells were collected. After washing with sterile water, the cells were suspended in 5 ml of Solution I (2 mM glucose, 10 mM EDTA, 25 mM Tris-HCl (pH8.0)), 25 mg of lysozyme was added, and the mixture was allowed to stand at 0 ° C for 30 minutes. Add 10 ml of Solution II (1N-NaOH, 5% SDS), 0 ℃, 5
After leaving it to stand for 7.5 minutes, 7.5 ml of Solution III (3M sodium acetate (pH 4.8)) was added, and the mixture was left to stand at 0 ° C. for 30 minutes. Centrifuge this, add 50 ml of ethanol to the supernatant, further centrifuge to remove the supernatant and remove 5 ml of solution IV (10 mM sodium acetate,
50 mM Tris-HCl (pH 8.0) and Ribonuclease A solution (10 mg /
(2.5 ml) was added and left at room temperature for 20 minutes. Add 12 ml of ethanol to this and collect the plasmid by centrifugation, 70%
It was washed with ethanol, dried, and then dissolved in 0.4 ml of sterilized water.
The recombinant plasmid thus obtained was named pAN1.

(5) Determination of nucleotide sequence of insert fragment pAN1 obtained in (4) above was cleaved with a plurality of restriction enzymes,
A restriction enzyme map was prepared (see FIG. 1), and subcloning was performed by a conventional method. That is, as a restriction enzyme
Using HindIII, NdeI, and SacI, pAN1 was partially cleaved, and the plasmid in which the cleavage site was religated with T4 DNA ligase was transformed again into Escherichia coli DH5α, and a plurality of plasmids were prepared in the same manner as in (4) above. These are the primer fluorescent labeling automatic DNA sequencer (LI-COL, model4000
The nucleotide sequence of the inserted fragment was analyzed by L). as a result,
A 2,208 bp gene sequence (SEQ ID NO: 1) and a 736 deduced amino acid sequence (SEQ ID NO: 2) were obtained.
It was named ga36A.

Next, enzymes having homology were searched for in the DDBJ (DNA Data Bank of Japan) database. This enzyme is an enzyme that is classified into family 36 of glycolytic enzymes: clan GH-D, and contains Bacillus halodurans.
us halodurans) BH2223 with about 57% homology, Bacillus
Bacillus stearothermophilu
52% homology with AgaN of s), about 57% homology with the same strain GalA,
Lactbacillus plantar
um) 43% homology with MelA, Pediococcus pentosaceus Aga1 with 42% homology, Aga2 with 35% homology, Streptococcus mutans Aga with It showed 37% homology (see Figures 2 to 4).

[0050]

EFFECT OF THE INVENTION By using α-galactosidase derived from Clostridium stercorarium, it is possible to carry out sugar conversion at a higher temperature.

[0051]

[Sequence list]                                SEQUENCE LISTING         <110> Mitsubishi Rayon Co., Ltd. <120> Thermostable α-galactosidase gene <130> P140192000 <140> <141> <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 2208 <212> DNA <213> Clostridium stercorarium <400> 1 atgccaatta tttttcatga agaatctggt gaatttcatc tttataataa aagcatcagc 60 tatataatta aaatattgcc taataaacag cttggaaacc tttatttcgg caaagttata 120 aaagacagaa agtcattttc acatttattc cagaaaaaag ccagaccctt atcggcttat 180 gtttttgaag atgatccggc tttttcactt cagcatacca tgcaagaata tccgtcatat 240 gggactactg attttagaag ccctgctttt gagataaagc agaaaaatgg gagcagaata 300 tcctgttttg aatataaaaa acatgaaatt tttgcaggca agaatggctt agaaggcctt 360 ccggcgactt atgtggaaga accagccgat gcagataccc ttgaaataac gctgtatgat 420 gaacttatta aaacagaact ggttttgagc tatacaatat tcaacaatta ttctgcgata 480 gccagaagcg caaggttcat aaacaaagga gaagaaacga ttgtgctgga aagggcaatg 540 agcgcaagca ttgattttat tgacagcgat tttgagatga tccagctttc cggcgcatgg 600 tcacgggaac ggcatattaa aacgagaaaa cttcagcagg gcatacaggg ggtttacagt 660 atgcgcggta ttagcagtgc ggaacataat ccgtttattg ccctaaaaag accaaatacg 720 gatgaaaaca gcggcgaagt atatggtttc agcttaatat acagcggcaa ccatcttgaa 780 caggttgagg tagataccca caacatgaca agagttatgc tgggtattca tccggatact 840 tttgagtggc cgttggaaaa aggagaattt tttcagacac ctgaagcaat aattgtttat 900 tcagacgagg ggctcaataa gatgagtcag acattccata agctgttcag caaacatctt 960 ataagcagac aatggcgtga caaacccaga ccggttctta taaacagttg ggaagctact 1020 ggcgcaaatt ttactgagga aaagctttta agaatggcag aaatagcaaa ggaacttgga 1080 atcgaactgt ttgtgcttga tgacggatgg tttggaaacc gagacaacga taaagcaggg 1140 ctgggagatt ggaatattgt taacaggaaa aaacttcccg atggtattga aggcctggct 1200 gacaaaattg aatcaatggg catgaaattc gggctttggt ttgagccgga atcagtaaat 1260 aaggacagca acttatacag aaaacaccct gattggataa ttgcgacccc gggaagaagg 1320 acatcagctt cacgtaacca gtacgttttg gatttctcga gaaaagaagt ggttgattat 1380 atttattccg taatggaaaa agttctgagc agtgcaaaaa tatcctatgt aaaatgggat 1440 atgaacaggt atataactga gtgttattct ttgggggcgg aagccggcag ccaaggaacg 1500 gtttttcacc ggtatattct gggagtatat aatttatttt caagattgac agagaggttt 1560 ccatatatac tttttgaagc atgctcaagc ggtggggcac ggtttgatcc cggaatactg 1620 tatttatctc cgcaagtatg gacaagcgat aatactgatg caattgagag actgaagatt 1680 cagtatggta cttctctgct ctatccaata agtactatgg gagctcatgt ttcagaagtt 1740 cccaatcagc aagtaggaag aattacgcct attgaaacaa gagccaatgt agcttatttc 1800 ggagtgtttg gttatgaact tgatttgact ttattaacag aagaagaaaa agaaaagata 1860 aaaaaacaga ttacttttta taaattccac agagaacttt ttcttaaagg aacattttac 1920 cgtatattaa gcccgtttga cggaaatgaa acgtcctgga tagttgtatc agaagataaa 1980 tcagaagcta ttgcagcgta ttacaagact ctgaattatg ctaacaaagg ctggaaaagg 2040 cttaaacttg tagggcttga cagagataag aaatatatta ttgacaacga caatgaaagg 2100 tattattacg gtgatgaact tatgaatgtc ggtattgtat taaaggatga agaattatgt 2160 gcaaacggaa atgacttttc gtccattatc ttttatttga aaagtatc 2208 <210> 2 <211> 736 <212> PRT <213> Clostridium stercorarium <400> 2 Met Pro Ile Ile Phe His Glu Glu Ser Gly Glu Phe His Leu Tyr Asn   1 5 10 15 Lys Ser Ile Ser Tyr Ile Ile Lys Ile Leu Pro Asn Lys Gln Leu Gly              20 25 30 Asn Leu Tyr Phe Gly Lys Val Ile Lys Asp Arg Lys Ser Phe Ser His          35 40 45 Leu Phe Gln Lys Lys Ala Arg Pro Leu Ser Ala Tyr Val Phe Glu Asp      50 55 60 Asp Pro Ala Phe Ser Leu Gln His Thr Met Gln Glu Tyr Pro Ser Tyr  65 70 75 80 Gly Thr Thr Asp Phe Arg Ser Pro Ala Phe Glu Ile Lys Gln Lys Asn                  85 90 95 Gly Ser Arg Ile Ser Cys Phe Glu Tyr Lys Lys His Glu Ile Phe Ala             100 105 110 Gly Lys Asn Gly Leu Glu Gly Leu Pro Ala Thr Tyr Val Glu Glu Pro         115 120 125 Ala Asp Ala Asp Thr Leu Glu Ile Thr Leu Tyr Asp Glu Leu Ile Lys     130 135 140 Thr Glu Leu Val Leu Ser Tyr Thr Ile Phe Asn Asn Tyr Ser Ala Ile 145 150 155 160 Ala Arg Ser Ala Arg Phe Ile Asn Lys Gly Glu Glu Thr Ile Val Leu                 165 170 175 Glu Arg Ala Met Ser Ala Ser Ile Asp Phe Ile Asp Ser Asp Phe Glu             180 185 190 Met Ile Gln Leu Ser Gly Ala Trp Ser Arg Glu Arg His Ile Lys Thr         195 200 205 Arg Lys Leu Gln Gln Gly Ile Gln Gly Val Tyr Ser Met Arg Gly Ile     210 215 220 Ser Ser Ala Glu His Asn Pro Phe Ile Ala Leu Lys Arg Pro Asn Thr 225 230 235 240 Asp Glu Asn Ser Gly Glu Val Tyr Gly Phe Ser Leu Ile Tyr Ser Gly                 245 250 255 Asn His Leu Glu Gln Val Glu Val Asp Thr His Asn Met Thr Arg Val             260 265 270 Met Leu Gly Ile His Pro Asp Thr Phe Glu Trp Pro Leu Glu Lys Gly         275 280 285 Glu Phe Phe Gln Thr Pro Glu Ala Ile Ile Val Tyr Ser Asp Glu Gly     290 295 300 Leu Asn Lys Met Ser Gln Thr Phe His Lys Leu Phe Ser Lys His Leu 305 310 315 320 Ile Ser Arg Gln Trp Arg Asp Lys Pro Arg Pro Val Leu Ile Asn Ser                 325 330 335 Trp Glu Ala Thr Gly Ala Asn Phe Thr Glu Glu Lys Leu Leu Arg Met             340 345 350 Ala Glu Ile Ala Lys Glu Leu Gly Ile Glu Leu Phe Val Leu Asp Asp         355 360 365 Gly Trp Phe Gly Asn Arg Asp Asn Asp Lys Ala Gly Leu Gly Asp Trp     370 375 380 Asn Ile Val Asn Arg Lys Lys Leu Pro Asp Gly Ile Glu Gly Leu Ala 385 390 395 400 Asp Lys Ile Glu Ser Met Gly Met Lys Phe Gly Leu Trp Phe Glu Pro                 405 410 415 Glu Ser Val Asn Lys Asp Ser Asn Leu Tyr Arg Lys His Pro Asp Trp             420 425 430 Ile Ile Ala Thr Pro Gly Arg Arg Thr Ser Ala Ser Arg Asn Gln Tyr         435 440 445 Val Leu Asp Phe Ser Arg Lys Glu Val Val Asp Tyr Ile Tyr Ser Val     450 455 460 Met Glu Lys Val Leu Ser Ser Ala Lys Ile Ser Tyr Val Lys Trp Asp 465 470 475 480 Met Asn Arg Tyr Ile Thr Glu Cys Tyr Ser Leu Gly Ala Glu Ala Gly                 485 490 495 Ser Gln Gly Thr Val Phe His Arg Tyr Ile Leu Gly Val Tyr Asn Leu             500 505 510 Phe Ser Arg Leu Thr Glu Arg Phe Pro Tyr Ile Leu Phe Glu Ala Cys         515 520 525 Ser Ser Gly Gly Ala Arg Phe Asp Pro Gly Ile Leu Tyr Leu Ser Pro     530 535 540 Gln Val Trp Thr Ser Asp Asn Thr Asp Ala Ile Glu Arg Leu Lys Ile 545 550 555 560 Gln Tyr Gly Thr Ser Leu Leu Tyr Pro Ile Ser Thr Met Gly Ala His                 565 570 575 Val Ser Glu Val Pro Asn Gln Gln Val Gly Arg Ile Thr Pro Ile Glu             580 585 590 Thr Arg Ala Asn Val Ala Tyr Phe Gly Val Phe Gly Tyr Glu Leu Asp         595 600 605 Leu Thr Leu Leu Thr Glu Glu Glu Lys Glu Lys Ile Lys Lys Gln Ile     610 615 620 Thr Phe Tyr Lys Phe His Arg Glu Leu Phe Leu Lys Gly Thr Phe Tyr 625 630 635 640 Arg Ile Leu Ser Pro Phe Asp Gly Asn Glu Thr Ser Trp Ile Val Val                 645 650 655 Ser Glu Asp Lys Ser Glu Ala Ile Ala Ala Tyr Tyr Lys Thr Leu Asn             660 665 670 Tyr Ala Asn Lys Gly Trp Lys Arg Leu Lys Leu Val Gly Leu Asp Arg         675 680 685 Asp Lys Lys Tyr Ile Ile Asp Asn Asp Asn Glu Arg Tyr Tyr Tyr Gly     690 695 700 Asp Glu Leu Met Asn Val Gly Ile Val Leu Lys Asp Glu Glu Leu Cys 705 710 715 720 Ala Asn Gly Asn Asp Phe Ser Ser Ile Ile Phe Tyr Leu Lys Ser Ile                 725 730 735

[0052]

[Brief description of drawings]

FIG. 1 shows a restriction enzyme map of pAN1 and its derivatives (pANS1 to pANS7). The large white arrow is aga36A and ORF2
Position and orientation, with small arrows indicating sequencing length and orientation.

FIG. 2 shows the results of searching for enzymes having homology by DDBJ (DNA Data Bank of Japan). In the figure, Cs is Clostridium strecorarium of the present invention, Bh is B. haloduran
s and Bs are B. stearothermophilus, Lp is L. plantaru
m and Pp are P. pentosaceus and Sm is S. mutans.

FIG. 3 shows a sequence continued from FIG.

FIG. 4 shows a continuation of FIG.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/40 C12N 5/00 AF term (reference) 4B024 AA03 AA05 BA12 CA03 DA06 EA04 GA11 GA27 4B050 CC01 CC03 DD02 EE01 LL02 LL05 4B065 AA23X AA23Y AA26X AA58X AA72X AA87X AB01 AC14 BA02 BA21 BA24 BB01 BC01 BC50 BD01 CA31 CA41

Claims (7)

[Claims] 1. A recombinant protein according to the following (a) or (b): (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid has been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and α- Protein with galactosidase activity 2. A gene encoding the following recombinant protein (a) or (b). (a) a protein containing the amino acid sequence represented by SEQ ID NO: 2 (b) consisting of an amino acid sequence in which at least one amino acid has been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and α- Protein with galactosidase activity 3. A gene containing the following DNA (c) or (d): (c) a DNA containing the nucleotide sequence represented by SEQ ID NO: 1 (d) a protein which hybridizes with the DNA containing the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions and which has α-galactosidase activity DNA encoding 4. A recombinant vector containing the gene according to claim 2 or 3. 5. A transformant containing the recombinant vector according to claim 4. 6. A method for producing the protein, which comprises culturing the transformant according to claim 5 in a medium and collecting a protein having an α-galactosidase activity from the resulting culture. 7. A method for producing a protein, which comprises culturing a bacterium belonging to Clostridium stercoralium in a medium and collecting a protein having an α-galactosidase activity from the obtained culture.