JP3132913B2 - L-fucose dehydrogenase gene, novel recombinant DNA and method for producing L-fucose dehydrogenase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an L-fucose dehydrogenase gene, a novel recombinant DNA and a method for producing L-fucose dehydrogenase (hereinafter abbreviated as L-FDH). L-FDH is an enzyme that catalyzes the reaction of oxidizing L-fucose to L-fuconolactone and reducing NADP to NADPH in the presence of L-fucose and NADP, as shown by the following formula.

L-fucose + NADP → L-fuconolactone + NADPH + H ⁺ L-FDH is extremely useful as an enzyme for quantifying L-fucose in L-fucose-containing samples such as serum.

[0003]

2. Description of the Related Art Conventionally, L-FDH, for example, belongs to the genus Pseudomonas and has a L-FDH-producing ability using NADP as a coenzyme is cultured in a medium, and L-FDH is collected from the culture. It is manufactured by.
However, the above-mentioned production method was not always satisfactory in terms of the yield of L-FDH and the like.

[0004]

The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, have found that LF
Succeeded in determining the structure of the DH gene,
Obtaining a recombinant DNA in which a DNA containing a gene encoding FDH is inserted into a vector DNA; introducing the recombinant DNA into a strain belonging to the genus Escherichia; culturing a microorganism having an L-FDH-producing ability in a medium; Thus, the present inventors have found that L-FDH is efficiently produced, and completed the present invention.

[0005]

The present invention resides in an L-fucose dehydrogenase gene encoding the amino acid sequence of SEQ ID NO: 2. Furthermore, the present invention resides in a recombinant DNA comprising a vector DNA containing the L-fucose dehydrogenase gene.

Further, the present invention relates to the above-mentioned recombinant DNA.
Wherein the microorganism belonging to the genus Escherichia having the ability to produce L-fucose dehydrogenase is cultured in a medium, and L-fucose dehydrogenase is collected from the culture. Hereinafter, the present invention will be described in detail.

First, Pseudomonas sp. No. 11 may be used as a donor of the gene of the present invention.
43 (FERM BP-2056) and the like. Then, a strain of Pseudomonas sp. No. 1143 is cultured by a known method, and centrifuged to obtain cells. From this cell, for example, Current P
Chromosomal DNA is obtained by the method described in rotocols in Molecular Biology unit 2.4.

Next, this chromosomal DNA is transformed into, for example, Sau3AI
To obtain a mixture of chromosomal DNA fragments. From the DNA fragment mixture thus obtained, for example, by a conventional agarose gel electrophoresis method, preferably 10-20
After obtaining a DNA fragment mixture having a size of Kb and subjecting it to an alkaline phosphatase treatment and the like, the mixture is further purified by a purification means such as phenol extraction, and further concentrated by a means such as ethanol precipitation and purified. The obtained DNA fragment mixture (including a DNA fragment containing the gene encoding L-FDH) is obtained.

On the other hand, the vector DNA which can be used in the present invention includes, for example, bacteriophage vector DNA, plasmid vector DNA and the like, and specifically, λEMBL4 DNA (manufactured by STRATAGENE) is preferred. The above bacteriophage vector D
In order to be able to incorporate the Sau3AI fragment, NA is digested with, for example, the restriction enzymes BamHI and SalI (manufactured by Takara Shuzo Co., Ltd.),
Bacteriophage vector D capable of incorporating u3AI fragment
Obtain the NA fragment.

Next, the above Pseudomonas sp.
43 containing a gene encoding L-FDH
A fragment and the bacteriophage vector DNA capable of incorporating the Sau3AI fragment are mixed,
A recombinant phage DNA is obtained by the action of 4DNA ligase (Boehringer Mannheim). This recombinant phage DNA can form phage particles by a usual in vitro packaging method.
The phage can be infected with, for example, Escherichia coli P2392 [obtained from Toyobo Co., Ltd.] to obtain plaques of recombinant phage having various DNA fragments.

[0011] In this plaque, a search for a recombinant phage having a DNA fragment containing the L-FDH gene is performed by using, for example, an oligonucleotide labeled with [γ- ^32P ] ATP (obtained from Amersham Japan) as a probe.
Current Protocols in Molecular Biology unit 6.3
Plaque hybridization can be performed by the method described above.

From the thus obtained recombinant phage (containing a DNA fragment containing the L-FDH gene), for example, Molecular Cloning P.371-
372 (Cold Spring Harbor Laboratory, 1982) can be used to obtain purified phage DNA. The purified recombinant phage DNA contains a large amount of unnecessary DNA other than the gene encoding L-FDH. Therefore, the unnecessary DNA is removed by the following operation. In other words, L-
The FDH gene is digested with a restriction enzyme having no cleavage site on the FDH gene, for example, EcoRI to obtain a DNA fragment mixture. L is added to any of the DNA fragments in this DNA fragment mixture.
Whether or not the gene encoding FDH is contained is determined by Southern hybridization using the above-mentioned radioactive oligonucleotide probe. Next, for the DNA fragment mixture obtained by digestion as described above,
GENECLEAN II kit [obtained from Funakoshi Co., Ltd.] was obtained by performing normal agarose gel electrophoresis and determining only the DNA fragment containing the gene encoding L-FDH.
And purified EcoRI digested fragments or Aor
51HI (Takara Shuzo) and MluI (Takara Shuzo) digestion fragments
(A gene encoding L-FDH is contained therein.)

On the other hand, any vector DNA can be used in the present invention, such as plasmid vector DNA and bacteriophage vector D.
NA and the like, and specifically, for example, a plasmid
pUC118, pUC119, pBLUESCRIPT II (manufactured by STRATAGENE) D
NA and the like are preferred. The vector DNA is digested with a restriction enzyme such as EcoRI or SmaI (Takara Shuzo) to obtain a digested vector DNA.

Next, a DNA fragment containing the above-described L-FDH-encoding gene and a cut vector DNA
Are mixed, and the mixture is reacted with, for example, T4 DNA ligase (Boehringer Mannheim) to obtain a recombinant DNA. Using this recombinant DNA, for example, E. coli K-1
2, preferably E. coli JM109 (obtained from Takara Shuzo), XL1-Blu
e (obtained from Funakoshi) and the like to obtain the respective strains. This transformation was performed according to the method of DM Morrison (Method
s in Enzymology, vol. 68, 326-331, 1979).

Using the DNA containing the above-mentioned L-FDH gene, the entire nucleotide sequence of the L-FDH gene is analyzed by the method shown in item (5) of the following Examples, and then the gene having the above-mentioned nucleotide sequence is analyzed. The amino acid sequence of the polypeptide translated by is determined. Next, the above L-
A strain belonging to the genus Escherichia having the ability to produce FDH is cultured to produce L-FDH.

This cultivation may be carried out by a conventional solid culturing method or liquid culturing method, but preferably a liquid culturing method is used. Further, as a medium used in the culture, for example, yeast extract, peptone, meat extract, corn steep liquor or one or more nitrogen sources such as leachate of soybean or wheat koji, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, One or more inorganic salts such as magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added.

The initial pH of the medium is suitably adjusted to 7-9. The medium is preferably cultured at 30-42 ° C., preferably about 37 ° C., for 6-24 hours by aeration and agitation deep culture, shaking culture, static culture and the like. After completion of the culture, L-FDH can be collected from the culture using a conventional enzyme collecting means. L obtained as described above
-FDH has exactly the same physicochemical properties as L-FDH described in JP-A-2-84177.

[0018]

The present invention will be described in more detail with reference to the following examples. However, the technical scope of the present invention is not limited by these examples. Example 1. Preparation of chromosomal DNA of Pseudomonas sp. No. 1143 Pseudomonas sp. No. 1143 (FERM BP-2056) was used in a medium [0.1% Polypepton, 0.1% yeast extract, 0.09% KH ₂ PO].
_4, 0.11% K ₂ HPO _4, 0.05% MgSO ₄ 7H ₂ O and 0.001% FeSO ₄ 7H
₂ O, (pH 7.0)] and cultured with shaking at 30 ° C for 40 hours. The culture was centrifuged at 8000 rpm for 10 minutes to collect the cells, followed by Current Protocols in Molecular Biology (WILEY I
nterscience, 1989) Unit 2.4, about 100 μg of chromosomal DNA was obtained. 2. Preparation of chromosomal DNA library 100 µg of the above chromosomal DNA was partially digested with Sau3AI according to a conventional method, and then 10 µg of a DNA fragment of a 10 to 20 kb fraction was obtained by agarose gel electrophoresis. A BamHI digest of 1 μg of the bacteriophage vector EMBL4 DNA and 2 μg of the above-mentioned Pseudomonas sp.
k Gold, manufactured by Stratagene).
Infect E.coli P2 392 (obtained from Stratagene),
50,000 plaques were obtained. The obtained plaque is used as a nylon membrane filter Hybond-N ⁺ (manufactured by Amersham).
Was blotted according to the protocol of Amersham. 3. L-FDH by plaque hybridization
Isolation of gene DEAE- from the cultured cells of Pseudomonas sp.
Sephadex, L-FDH purified using Sephadex G-100 was purified by the method of Gross and Witkop (Journal of Biolo
gical Chemistry, 237, 1856, 1962), and treated with cyanogen bromide to fragment the peptide. This is reversed phase H
The fractions were separated by PLC, and the amino acid sequence was determined using a protein sequencer (Applied Biosystems, model 470A). Amino acid sequence obtained Ile Pro Ala Leu
Gly Tyr Gly Ala Ala Asn ValGly Asn Leu Phe polynucleotide ATICCIGCICTIGGITATGGIGCIGCIAAT
GTIGGIAATCTITT (probe F44) and amino acid sequence Me
t Val Ala Gly Arg TyrThr Leu Leu Glu Gln Pro Polynucleotide reverse chain GGTTGTTCIAGIAGIGTATAI
CGICCIGCIACCAT (probe F35) (A represents adenine, C represents cytosine, G represents guanine, T represents thymine, and I represents inosinic acid) was synthesized using a DNA synthesizer (Applied Biosystems, Model 392). 1 μg of this synthetic DNA was end-labeled with [γ- ³² P] ATP (Amersham) and T4 polynucleotide kinase (Takara Shuzo) to use as a probe for plaque hybridization. Current Protocols in Molecular Biology unit 6.
Plaque hybridization was performed according to the method described in 3, and three positive clones were obtained. 4. Analysis of L-FDH gene The phage DNA of the three positive clones isolated was subjected to a conventional method (Molecular Cloning, ed. Maniatis et al., P.85, C
old Spring Harbor Laboratory, USA, 1982), and Southern hybridization (J. Mol. Biol., 98,
503, 1975).
It was found that the 0.55 kb DNA fragment hybridized with the probe commonly in the three strains. Therefore, for one of these three strains, the EcoRI fragment containing the PstI fragment was prepared by agarose gel electrophoresis, and then prepared by GENECLEAN II (purchased from Funakoshi).
Inserted into UC119 DNA. Next, Aor51HI (purchased from Takara Shuzo) containing the L-FDH gene was inserted from the inserted fragment.
A 1.5 kb DNA fragment sandwiched between MluI (purchased from Takara Shuzo) was prepared by the above-described method, and further, a DNA Blunting kit (purchased from Takara Shuzo).
After blunting the ends using Takara Shuzo, pBLUESCRIPT II SK (STRATAGEN) cut with SmaI (purchased from Takara Shuzo)
(Purchased from E company) and insert the recombinant plasmid pFD203
DNA was prepared.

FIG. 1 shows a map of the cleavage of pFD203 by restriction enzymes. 5. Determination of nucleotide sequence The nucleotide sequence of pFD203 was determined using a deletion kit for Kilo-Sequence (purchased from Takara Shuzo) and a 370 DNA Sequencing System (purchased from Applied Biosystems). The determined nucleotide sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the polypeptide translated from the gene having the nucleotide sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. The L-FDH gene had a coding region of 987 bases and encoded 329 amino acids. 6. Expression of L-FDH gene in Escherichia coli Recombinant plasmid pFD203 DNA prepared in 4 above
DM Morri-son method (Methods in Enzymol
ogy, 68, 326, 1979), transforming Escherichia coli XL1-Blue, transformant, E. coli recombinant XL1-Blue (pFD203)
I got Escherichia coli (E. coli) XL1-Blue (pFD203) was supplied to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology.
(FERM BP-3996).

The E. coli XL1-Blue (p
FD203) with 1 mM IPTG (isopropyl-β-D-galactoside)
TY medium containing 1% Bacto-trypton, 0.5% Bacto-yeas
t extract, 0.5% NaCl) in 10 ml at pH 7.2 at a temperature of 37 ° C.
The cells were cultured for 16 hours. The L-FDH activity in the culture was determined by
As a result of measurement by the method described in -84177, 0.1 U / ml
Met.

[0021]

According to the present invention, an L-fucose dehydrogenase gene and a recombinant DNA containing the same are provided, and by culturing a microorganism belonging to the genus Escherichia containing the recombinant DNA and having an ability to produce L-fucose dehydrogenase. , L-FDH could be produced efficiently. Therefore, the present invention is industrially very useful.

[0022]

[Sequence list]

 1. SEQ ID NO: 1 (1) Sequence length: 987 (2) Sequence type: nucleic acid (3) Number of strands: double-stranded (4) Topology: linear (5) Sequence type: chromosomal DNA (6) origin: organism: Pseudomonas sp.No.1143 (7) SEQ: ATGTCATCGA CTGAGCCTGC CGCGGCCGCT GCCGGCCTGG CCATCCCGGC CCTCGGCTAC 60 GGCGCCGCCA ACGTGGGCAA CCTCTTCCGC GCGCTGAGCG ATGACGAGGC CTGGGCCGTG 120 CTCGAGGCGG CGTGGGATGC CGGCATCCGC TATTACGACA CCGCGCCGCA CTACGGGCTC 180 GGGCTGAGCG AGAAGCGCCT CGGTGCCTTC CTGCAGACCA AGCCGCGCGA CGAGTTCGTC 240 GTGTCGACCA AGGCCGGGCG CCTGCTGCGC CCGAACCCCG AGCGCCGGCC GAGCGGCCTG 300 GACACCGACA ACGACTTCCA CGTGCCCGAC GACCTGCGCC GCGAGTGGGA CTTCACCGAG 360 CAGGGCATCC GTGCGAGCAT CGCCGAGTCG CAGGAGCGGC TCGGGCTCGA CCGCATCGAC 420 CTGCTGTACC TGCACGACCC CGAGCGGCAC GACCTCGACC TCGCGCTCGC CTCGGCCTTC 480 CCGGCGCTCG AGAAGGTGCG CGCCGAGGGC GTCGTGAAGG CGATCGGCAT CGGCTCGATG 540 GTGTCGGATG CCCTGACCCG GGCGGTCCGC GAGGCGGACC TCGACCTCAT CATGGTCGCC 600 GGGCGCTACA CGCTGCTCGA GCAGCCCGCG GCCA CCGAGG TGCTGCCTGC ATGCGCTGAG 660 AACGCCACCG GGATCGTCGC GGCATCCGTC TTCAATTCCG GACTGCTCGC GCAGAGCGAG 720 CCGAAGCGCG ACGGACGCTA CGAGTACGGT CAGCTGCCCG ATGAGCTGTG GGATCGCCTG 780 GTGCGGATCG CCGCGATCTG CCGCAACCAC GACGTACCGC TGCCGGCGGC GGCGATCCAG 840 TTCCCGCTGC AGTCGGCCCT GGTGCGCTCG GTCGTGGTCG GCGGCAGCCG CCCCGCGCAG 900 CTGACGCAGA ACGCCGAGTA CGCCGCGCTC GAGATCCCCG CCGGGCTGTG GGCCGAGCTG 960 GCCGAGGCCC GCCTGATCCC CACCCCC 987 2. SEQ ID NO: 2 (1) Sequence length: 329 (2) Sequence type: amino acid (3) Topology: linear (4) Sequence type: protein (5) Origin: Organism name: Pseudomonas sp. No. 1143 (6) Sequence: Met Ser Ser Thr Glu Pro Ala Ala Ala Ala Ala Ala Gly Leu Ala Ile 15 Pro Ala Leu Gly Tyr Gly Ala Ala Asn Val Gly Asn Leu Phe Arg 30 Ala Leu Ser Asp Asp Glu Ala Trp Ala Val Leu Glu Ala Ala Trp 45 Asp Ala Gly Ile Arg Tyr Tyr Asp Thr Ala Pro His Tyr Gly Leu 60 Gly Leu Ser Glu Lys Arg Leu Gly Ala Phe Leu Gln Thr Lys Pro 75 Arg Asp Glu Phe Val Val Ser Thr Lys Ala Gly Arg Leu Leu Arg 90 Pro Asn Pro Glu Arg Arg Pro Ser Gly Leu Asp Thr Asp Asn Asp 105 Phe His Val Pro Asp Asp Leu Arg Arg Glu Trp Asp Phe Thr Glu 120 Gln Gly Ile Arg Ala Ser Ile Ala Glu Ser Gln Glu Arg Leu Gly 135 Leu Asp Arg Ile Asp Leu Leu Tyr Leu His Asp Pro Glu Arg His 150 Asp Leu Asp Leu Ala Leu Ala Ser Ala Phe Pro Ala Leu Glu Lys 165 Val Arg Ala Glu Gly Val Val Lys Ala Ile Gly Ile Gly Ser Met 180 Va l Ser Asp Ala Leu Thr Arg Ala Val Arg Glu Ala Asp Leu Asp 195 Leu Ile Met Val Ala Gly Arg Tyr Thr Leu Leu Glu Gln Pro Ala 210 Ala Thr Glu Val Leu Pro Ala Cys Ala Glu Asn Ala Thr Gly Ile 225 Val Ala Ala Ser Val Phe Asn Ser Gly Leu Leu Ala Gln Ser Glu 240 Pro Lys Arg Asp Gly Arg Tyr Glu Tyr Gly Gln Leu Pro Asp Glu 255 Leu Trp Asp Arg Leu Val Arg Ile Ala Ala Ile Cys Arg Asn His 270 Asp Val Pro Leu Pro Ala Ala Ala Ile Gln Phe Pro Leu Gln Ser 285 Ala Leu Val Arg Ser Val Val Val Gly Gly Ser Arg Pro Ala Gln 300 Leu Thr Gln Asn Ala Glu Tyr Ala Ala Leu Glu Ile Pro Ala Gly 315 Leu Trp Ala Glu Leu Ala Glu Ala Arg Leu Ile Pro Thr Pro 329

[Brief description of the drawings]

FIG. 1 is a diagram showing a map of cleavage of a recombinant plasmid pFD203 DNA by a restriction enzyme.

────────────────────────────────────────────────── ─── Continued on front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 9/04 C12R 1:38) (C12N 15/09 ZNA C12R 1:38) (72) Inventor Tatsuo Horiuchi Noda City, Chiba Prefecture Noda 399 Noda Institute of Scientific and Industrial Research (72) Inventor Eiichi Nakano 339 Noda, Noda, Chiba Pref. Kikkoman Corporation (56) References Agric. Biol. Chem. , 53 (6), p. 1493-p. 1501, (1989) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00 C12N 1/21 C12N 9/04 GenBank / EMBL / DDBJ (GENETYX) MEDLINE (STN)

Claims (3)

(57) [Claims] 1. An L-fucose dehydrogenase gene encoding the amino acid sequence of SEQ ID NO: 2. 2. A recombinant DNA comprising a vector DNA containing the L-fucose dehydrogenase gene according to claim 1. 3. A microorganism belonging to the genus Escherichia containing the recombinant DNA according to claim 2 and having an ability to produce L-fucose dehydrogenase, is cultured in a medium, and L-fucose dehydrogenase is collected from the culture. A method for producing L-fucose dehydrogenase.