JP2002360259A - New alkali phosphatase derived from shewanella sp. sib1 strain - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new polypeptide having alkali phosphatase enzyme activity and a new gene encoding the polypeptide. SOLUTION: New alkali phosphatase derived from Shewanella sp. SIB1 strain is provided by the present invention. Further, a polypeptide specifying the alkali phosphatase and a gene encoding the polypeptide are provided by the present invention. The enzyme of the present invention is especially useful as a reagent used in the field of genetic engineering, because the enzyme has characteristics effective in removing phosphoric acid group of 5' end of nucleic acid and capable of readily deactivating the enzyme by heat treatment after reaction because of low heat resistance.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Shewanella sp. S
The present invention relates to a novel alkaline phosphatase derived from IB1 strain.
Further, the present invention relates to a polypeptide that defines the alkaline phosphatase, and a gene encoding the polypeptide. Furthermore, the present invention relates to a method of using the enzyme as a reagent for dephosphorylating nucleic acids.

[0002]

2. Description of the Related Art Alkaline phosphatase
sphatase) is an enzyme that catalyzes the dephosphorylation of DNA fragments. This enzyme is widely distributed in animal tissues, and alkaline phosphatase activity in serum is derived from liver, small intestine, and bone tissue. I have. Alkaline phosphatase is also a typical periplasmic enzyme in E. coli.

[0003] Alkaline phosphatase, which catalyzes a dephosphorylation reaction, is widely used as a research reagent in the fields of biochemistry and molecular biology. For example,
Alkaline phosphatase is widely used in enzyme immunoassays, and is used as a labeling enzyme for antibodies to detect antigen-antibody reactions. Alkaline phosphatase is also used as a reagent for genetic engineering. As an example, when labeling the 5 'end of a nucleic acid with ³² P,
Alkaline phosphatase is also used as a reagent used in the pretreatment. That is, after removing the phosphate group at the terminal of the nucleic acid using alkaline phosphatase, the nucleic acid is reacted with [γ- ³² P] ATP to obtain the 5 ′
It is common practice to label the ends. The nucleic acid labeled in this manner can be used, for example, as a probe or the like.

[0004] In addition, when an operation of incorporating a heterologous DNA into a plasmid vector is carried out, in order to prevent self-ligation of the plasmid DNA, a treatment with alkaline phosphatase to remove the phosphate group at the 5 'end is performed. Have been done. In this case, it is necessary to remove the alkaline phosphatase activity after incorporation of a heterologous DNA so that the subsequent operation does not affect the alkaline phosphatase activity. Escherichia coli-derived alkaline phosphatase (BAP) has been used to remove the phosphate group at the 5 'end.
However, since BAP is extremely stable to heat, a complicated operation such as phenol / chloroform extraction is required to remove the enzyme activity after the reaction. The removal of the phosphate group at the 5 'end of plasmid DNA by alkaline phosphatase treatment will be described in detail later.

[0005]

Therefore, a novel alkaline phosphatase which is less stable to heat treatment than BAP is required as a reagent for removing the 5′-terminal phosphate group of nucleic acids such as DNA. Had been. By using alkaline phosphatase with low thermostability, the alkaline phosphatase used after the enzymatic reaction can be inactivated and the post-treatment is easy, so such alkaline phosphatase is useful as a reagent for research in genetic engineering. It is.

[0006]

Means for Solving the Problems One aspect of the present invention is as follows.
It is a novel protein derived from Shewanella sp. SIB1 strain and has the following properties. (1) under alkaline conditions, catalyze a hydrolysis reaction using a phosphoric acid monoester as a substrate to produce inorganic phosphoric acid;
(2) molecular weight is 49 kDa; (3) isoelectric point is 4.9; (4) optimal temperature of alkaline phosphatase activity is in the range of 30 ° C. to 40 ° C .; 5) At 70 ° C, the alkaline phosphatase activity disappears within 10 minutes.

Another aspect of the present invention is a polypeptide derived from Shewanella sp. SIB1 strain having alkaline phosphatase activity, wherein the polypeptide comprises an amino acid sequence represented by the following (a) or (b): And (A) amino acid numbers 1-455 shown in SEQ ID NO: 1 in the sequence listing
A polypeptide comprising an amino acid sequence represented by the formula: (B) producing an inorganic phosphoric acid by catalyzing a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions;
A polypeptide having an alkaline phosphatase activity, wherein a part of (a) is deleted, substituted or added.

[0008] A further aspect of the present invention is a gene encoding a polypeptide derived from Shewanella sp. SIB1 strain having alkaline phosphatase activity, which gene has the following nucleotide sequence (c) or (d): It is characterized by the following. (C) a gene comprising a nucleotide sequence represented by nucleotide numbers 1-1368 shown in SEQ ID NO: 2 of the sequence listing. (D) generating an inorganic phosphoric acid by catalyzing a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions;
A gene encoding a polypeptide having an alkaline phosphatase activity, wherein part (c) is deleted, substituted or added.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In general, the optimal reaction temperature of an enzyme produced by a psychrophilic bacterium such as a psychrophilic bacterium or a psychrotrophic bacterium shifts to a lower temperature than the optimal reaction temperature of an enzyme derived from a normal-temperature bacterium. Is known to be. Also, the thermal stability of these enzymes is generally significantly lower than that of enzymes derived from cold organisms. The present inventors have paid attention to the characteristics of such psychrophilic enzymes, and have attempted to collect alkaline phosphatase that can be easily inactivated by heat treatment. The present inventors have found that the psychrotrophic Shewanella s that can grow even at 0 ° C from domestic oil storage tanks.
p. SIB1 strain has been successfully isolated [Kato, T.,
et al. (2001) Appl. Microbiol. Biotech. in press.
]. Therefore, the gene encoding alkaline phosphatase was cloned from this bacterium, expressed in Escherichia coli, and its characteristics were analyzed. As a result, they found that alkaline phosphatase derived from the present bacterium was significantly more unstable to heat than alkaline phosphatase (BAP) derived from Escherichia coli.

Therefore, it is expected that alkaline phosphatase derived from the psychrotrophic Shewanella sp. SIB1 strain can be easily inactivated by heat treatment alone without performing complicated operations such as phenol / chloroform extraction. Alkaline phosphatase is an enzyme that plays an important role in the field of genetic engineering research as described in detail below, and alkaline phosphatase from Shewanella sp. Strain SIB1 provides a very useful tool in genetic engineering research. I think that the.

A heterologous DN of interest in a plasmid vector
In incorporating A, a plasmid and a heterologous DNA are each treated with a restriction enzyme to create a specific cleavage site (restriction site). Then, utilizing the specificity of the prepared cleavage site, the heterologous DNA can be incorporated into the plasmid vector by binding the plasmid and the heterologous DNA with DNA ligase. Such a method is the most common experimental operation performed when performing a genetic recombination experiment.

[0012] If the restriction site thus prepared is left as it is, self-ligation of the plasmid vector occurs, and the efficiency of integration of heterologous DNA is reduced. Therefore, a heterologous plasmid vector
Before incorporation of DNA, alkaline phosphatase
It is common practice to remove the phosphate group at the 5 'end of the plasmid vector to prevent self-ligation of the plasmid vector. For such a purpose, alkaline phosphatase (BAP) derived from Escherichia coli is currently widely used.

After removing the terminal phosphate group with alkaline phosphatase, if the enzyme activity of the alkaline phosphatase remains, it is necessary to deactivate the enzyme activity since it affects the subsequent operation. . BAP currently used is difficult to inactivate by heating because of its high thermal stability as described above, and phenol /
It is necessary to use a complicated means to remove the enzymatic activity, for example, by extracting with chloroform.

The alkaline phosphatase derived from the psychrotrophic Shewanella sp. SIB1 strain of the present invention has low thermostability.
Unlike the above BAP, the enzyme can be easily inactivated by heating. That is, there is no need to remove the enzyme by, for example, extracting with phenol / chloroform after the reaction, and the enzyme after the reaction can be inactivated by a simple heat treatment operation. It has the advantage of being easier. BAP, which is currently most widely used, does not decrease its enzyme activity even when treated at 70 ° C for 30 minutes. In comparison, as shown in the examples below, alkaline phosphatase derived from the psychrotrophic Shewanella sp. Strain SIB1 of the present invention almost completely inactivates alkaline phosphatase activity within 10 minutes at 70 ° C. . Therefore, alkaline phosphatase derived from Shewanella sp. SIB1 strain is remarkably low in thermostability and exhibits characteristics different from BAP, so that it can be used for various uses as a research reagent for genetic engineering.

As shown in the following examples, the Shewan of the present invention
Alkaline phosphatase derived from ella sp. SIB1 strain has the following enzymatic properties. (1) under alkaline conditions, catalyze a hydrolysis reaction using a phosphoric acid monoester as a substrate to produce inorganic phosphoric acid;
(2) molecular weight is 49 kDa; (3) isoelectric point is 4.9; (4) optimal temperature of alkaline phosphatase activity is in the range of 30 ° C. to 40 ° C .; 5) At 70 ° C, the alkaline phosphatase activity disappears within 10 minutes.

In the specification of the present application, alkaline phosphatase means an enzyme that catalyzes a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions to produce inorganic phosphate. Here, the alkaline condition means pH8.0-pH12.0, preferably pH9.0-pH11.0. As shown in the examples below, the optimal pH of the alkaline phosphatase of the present invention is 10.5.

The novel polypeptide having alkaline phosphatase activity and derived from the Shewanella sp. SIB1 strain of the present invention has the amino acid numbers 1-455 shown in SEQ ID NO: 1 in the sequence listing.
Specified by the amino acid sequence represented by This polypeptide is encoded by a gene specified by the nucleotide sequence set forth in SEQ ID NO: 2 in the sequence listing.

The polypeptide in which a part of the polypeptide shown in SEQ ID NO: 1 is deleted, substituted or added refers to 20 or less, preferably 10 or less, more preferably 5 or less in the amino acid sequence shown in SEQ ID NO: 1. A polypeptide in which no more than three amino acids have been substituted. In addition, such a polypeptide has 70% or more, preferably 80% or more, more preferably 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1. Such polypeptides are also within the scope of the present invention, as long as they have activity as alkaline phosphatase.

Further, the polypeptide encodes the above-mentioned polypeptide,
The novel gene derived from Shewanella sp. SIB1 strain is specified by the nucleotide sequence represented by nucleotide numbers 1-1368 shown in SEQ ID NO: 2 in the sequence listing. The nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing corresponds to the reading frame, and SEQ ID NO: 1 in the sequence listing.
Encodes the described polypeptide.

According to the genetic recombination technique, a specific portion of the basic DNA can be added without changing or improving the basic characteristics of the DNA.
Mutations can be caused artificially. A gene having a natural nucleotide sequence provided by the present invention, or a gene having a nucleotide sequence different from the natural one, can be similarly artificially inserted, deleted, and substituted to be equivalent to the natural gene. The present invention includes such mutant genes.

That is, the gene in which a part of the gene shown in SEQ ID NO: 2 is deleted, substituted or added is 20 or less, preferably 10 or less in the nucleotide sequence shown in SEQ ID NO: 2. Preferably, the gene has 5 or less bases substituted. Also, such a gene and SEQ ID NO: 2
Has a homology of 70% or more, preferably 80% or more, more preferably 90% or more. Such genes are also within the scope of the invention, as long as they encode a polypeptide having alkaline phosphatase activity.

A method for hydrolyzing a phosphate monoester in a nucleic acid such as DNA or RNA or a fragment thereof using the alkaline phosphatase derived from Shewanella sp. SIB1 of the present invention is also within the scope of the present invention. . The most typical use of this method is the use as a reagent for removing the 5′-terminal phosphate group of a plasmid DNA treated with a restriction enzyme as described above, but is not limited thereto, and is used as an alkaline phosphatase. Utilizing the enzyme activity, it can be used for various other uses.

[0023]

[Example] (Cloning of alkaline phosphatase gene) Plasmid pUC19 was digested with EcoRI,
The anella sp. SIB1 strain genome was cyclized by ligation with a fragment digested with EcoRI. Ligation was performed using a commercially available ligation kit (Takara) in accordance with the attached instructions. Escherichia coli strain JM109 was transformed with the thus obtained plasmid to obtain a large number of transformants. Plasmids were extracted from these transformants, and the nucleotide sequence of the inserted sequence was determined to find a fragment containing a part (5′-terminal portion) of the SIB1 strain-derived APase gene.

Next, Southern hybridization was carried out using this fragment as a probe DNA. First, SI
The fragment obtained by digesting the genome of the B1 strain with KpnI was subjected to 0.7% agarose electrophoresis and transferred to a nylon membrane. As a result of Southern hybridization using this membrane, the probe DNA hybridized to a DNA fragment of about 2.5 kb. Then, the fragment obtained by digesting the SIB1 strain genome with KpnI was subjected to 0.7% agarose electrophoresis,
A fragment with a length of around kb was purified. This fragment was cyclized by ligation of the plasmid pUC19 digested with KpnI. Escherichia coli strain JM109 was transformed with the thus obtained plasmid to obtain a large number of transformants. Colony hybridization was performed on these transformants, and plasmids were extracted from colonies to which the probe DNA had hybridized.

When the base sequence of the inserted sequence was determined for the obtained plasmid (pUC2500), alkaline phosphatase (Alkaline phosphatase: APase) derived from SIB1 strain was determined.
Gene full length. This gene was found to encode a protein consisting of 455 amino acid residues and having a molecular weight of 49 kDa and a pI of 4.9 (FIGS. 1 and 2). FIGS. 1 and 2 show a series of base sequences and amino acid sequences. The first half of the sequence is shown in FIG. 1, and the second half of the subsequent sequence is shown in FIG. The nucleotide sequence of the gene of the enzyme of the present invention is shown in the upper part of FIGS. 1 and 2 and in the sequence listing, SEQ ID NO: 2, and the deduced amino acid sequence is shown in the lower part of FIGS. Blast-based homology search with the database showed the highest homology (45%) with APase from Enterococcus faecalis. It also showed 36% homology with Escherichia coli APase known as BAP. From SIB1 strain
APase preserves all the active center residues revealed by the crystal structure of the Escherichia coli enzyme. Therefore, the catalytic mechanism of this enzyme is considered to be the same as that proposed for the Escherichia coli enzyme.

(Expression of APase gene) Usually, APase of Gram-negative bacteria is synthesized as a precursor, and when it moves into the periplasm, the N-terminal signal peptide is cleaved to become a mature form. Therefore, SignalP V2.0 World Wide Web Se
The cleavage site of the signal peptide was predicted by rver. Next, a protein (Asp28-Pro45) that is expected to be a mature APase
5) the gene encoding pET-25b vector (Novagen
) Was inserted immediately after the pelB leader sequence to construct an expression vector. The method is shown below (FIG. 3). First, the APase gene of pUC2500 was amplified by PCR using primers. As a primer, a chemically synthesized oligonucleotide shown in FIG. 4 was used. In FIG. 4, underlines indicate restriction enzyme sites. Plasmid pBR2500 as template DN
A and 5'-primer containing NcoI restriction enzyme site (1)
Approximately 1.3 kbp DNA fragment was amplified by PCR using 3′-primer (2) containing
A fragment was obtained. The primer (1) is shown in SEQ ID NO: 3 in the sequence listing, and the primer (2) is shown in SEQ ID NO: 4 in the sequence listing. PCR reaction is commercially available KOD polymerase (Toyobo)
And according to the instructions. The obtained fragment was digested with restriction enzymes NcoI and SalI, and the digest was purified by 0.7% agarose gel electrophoresis.

The pET-25b plasmid was digested with NcoI and SalI, a fragment of about 5.4 Kb was purified by 0.7% agarose electrophoresis, and ligated with the above-mentioned DNA fragment of about 1.3 kbp for cyclization. Escherichia coli JM109 strain was transformed with the thus obtained plasmid, and an APase gene expression plasmid pET-AP5 was obtained from the transformant. Using this plasmid, E. coli AC109, an APase-deficient strain, was used.
(DE3) strain was transformed. The resulting transformant was cultured at 37 ° C., and when the OD 600 reached about 0.5, IPTG was added to a final concentration of 1 mM, and the cells were further cultured for 5 hours. After collecting the cells, osmotic shock buffer (30 mM Tris
-HCl (pH 8.0), 1 mM EDTA, 20% sucrose) and left at room temperature for 10 minutes to apply osmotic shock. After centrifuging the suspension, the resulting precipitate was suspended in cold water and left on ice for 15 minutes. This suspension was centrifuged, and the obtained supernatant was used as a periplasm fraction. As a result, apparently strong activity was observed in the periplasm fraction, and SIB1-derived AP
Expression of ase was confirmed. APase activity uses p-nitrophenyl phosphate as a substrate and absorbs p-nitrophenol generated during the reaction at 410 nm (the molecular extinction coefficient is 1.61 x
10 ^{4 -1} cm ^-1 ). The reaction is performed with 1 ml of 50 ml containing 0.04% p-nitrophenyl phosphate.
mM Tris-HCl (pH 8.5) -1 mM MgCl ₂ -0.1 mM ZnCl ₂ -50 m
The reaction was performed at 30 ° C. for 15 minutes in M KCl and the reaction was stopped by adding 50 μl of 4 M NaOH.

Derived from SIB1 secreted by E. coli periplasm
Using the crude extract of APase, its optimal activity temperature (FIG. 5), optimal activity pH (FIG. 6), and thermal stability (FIG. 7) were measured. As a result, it was suggested that the optimum temperature of the SIB1-derived APase (black circle in FIG. 5) was shifted to a lower temperature side by about 20 ° C. than that of the E. coli enzyme (BAP: open circle in FIG. 5) (FIG. 5). Also,
The optimum pH of SIB1-derived APase was 10.5 (FIG. 6). FIG.
In, the open circles are values measured with glycine-NaOH buffer,
Filled circles indicate values measured with Tris-HCl. As can be seen from FIG. 6, since the optimal pH of BAP is reported to be 8-9, the optimal pH of SIB1-derived APase has shifted to a higher alkalinity than the optimal pH of BAP. After incubation of the enzyme solution at 70 ° C., the result of measuring the residual activity at 30 ° C. is shown in FIG. According to FIG. 7, SIB1-derived APase was almost completely inactivated by heat treatment at 70 ° C. for 5 minutes (black circles in FIG. 7), whereas BAP (open circles in FIG. 7) was extremely stable at this temperature (FIG. 7). 7). As a result, it was expected that the use of SIB1-derived APase would make the dephosphorylation operation easier than before.

[0029]

According to the present invention, a novel alkaline phosphatase derived from Shewanella sp. SIB1 strain has been provided. Further, according to the present invention, a polypeptide defining the alkaline phosphatase and a gene encoding the polypeptide are provided. The enzyme of the present invention is effective for removing the phosphate group at the 5 'end of nucleic acids, and has a characteristic that heat resistance is low, so that the enzyme can be easily inactivated by heat treatment after the reaction. It is particularly useful as a reagent used in the field of genetic engineering.

[0030]

[Sequence Table] <110> President of Osaka University <120> Novel alkaline phosphatase derived from Shewanella sp. SIB1 strain <160> 4 <210> 1 <211> 455 <212> amino acid <213> Shewanella sp. SIB1 strain <400 > 1 MHFLSTYQRI ISRLLLVSSL IVTGAMADEV IMPPAVANTA SNNVPTVGPS RPKNIIIMIG 60 DGMGPAYTTA YRYYKDNPDT EEIEQTVFDR LLVGMASTYP ASVSGYVTDS AASATALSTG 120 VKSYNGAIAV DTEKRPLTTL MEKAKALGLS TGVAVTSQVN HATPAAFLAH NESRSNYIDI 180 AEAYLTSDAD VILGGGQQYF TPTLLEQFSA KGYQHISEFS ELASVTTPKV LGLFADVQLP 240 WAINDKQAHK LSHMTQKALD LLSQNEQGFV LLVEGSLIDW AGHSNDIATA MGEMDEFASA 300 IEVVEQFVRQ SKDTLMVVTA DHNTGGLSVG AHDKYEWHPE VLHKVKASPD VIATRAIAED 360 NWQPLTATLL GFTPNEAQYS QLQSARMQGN EPLSIALRKL IDIQSNTGWT SGGHTAMDVQ 420 VFAEGPGARL FSGHQDNIDI AIKMFSLLPQ SVQTP 455 <210> 2 <211> 1368 <212> Nucleic acid <213> Shewanella sp. SIB1 strain <400> 2 ATGCATTTTT TATCTACCTA CCAACGGATA ATCAGCAGAT TATTATT GGT ATCTAGCTTG 60 ATCGTAACGG GGGCTATGGC TGATGAAGTC ATTATGCCGC CTGCAGTTGC TAATACAGCA 120 AGTAATAATG TACCAACTGT GGGTCCATCA AGACCTAAAA ATATCATCAT TATGATAGGT 180 GATGGTATGG GACCGGCTTA CACCACCGCC TATCGTTATT ATAAAGATAA TCCTGACACA 240 GAAGAGATTG AACAAACTGT GTTTGATCGC TTACTCGTTG GTATGGCAAG CACTTACCCA 300 GCTTCCGTCA GTGGTTATGT CACCGACTCT GCCGCATCTG CCACAGCACT GTCAACTGGA 360 GTTAAAAGTT ATAATGGTGC CATTGCCGTT GATACCGAAA AACGCCCACT CACAACCTTA 420 ATGGAAAAAG CCAAAGCATT AGGCTTGTCA ACGGGTGTAG CAGTTACATC CCAAGTAAAC 480 CATGCTACAC CTGCAGCATT TTTGGCTCAT AATGAAAGCC GTAGTAATTA TATAGATATT 540 GCTGAGGCTT ATTTAACATC TGATGCTGAT GTGATTCTTG GTGGTGGCCA GCAATATTTC 600 ACTCCAACAT TACTTGAGCA ATTTAGCGCT AAAGGCTACC AGCACATCAG TGAATTCTCT 660 GAGCTAGCAT CAGTGACTAC GCCAAAAGTA TTGGGGCTGT TTGCAGATGT ACAATTGCCT 720 TGGGCCATTA ATGATAAACA AGCGCATAAA CTCAGTCATA TGACCCAAAA GGCATTAGAT 780 TTACTGTCAC AAAATGAGCA AGGTTTTGTA TTGTTAGTTG AAGGCAGCTT AATTGATTGG 840 GCTGGCCACA GTAACGATAT TGCTACCGCT ATGGGTGAAA TGGATGAATT TGCATCCGCT 900 AT CGAAGTGG TTGAACAATT TGTTCGTCAA AGTAAAGACA CCTTGATGGT TGTGACAGCA 960 GATCATAATA CCGGTGGATT ATCAGTTGGC GCACATGACA AATACGAATG GCACCCTGAA 1020 GTACTGCATA AGGTTAAAGC CAGCCCTGAT GTCATTGCTA CCCGTGCTAT TGCCGAAGAC 1080 AATTGGCAGC CTTTAACGGC AACGCTTCTT GGCTTTACTC CAAATGAAGC GCAATACAGT 1140 CAATTACAAA GCGCACGCAT GCAAGGTAAT GAACCATTAT CAATCGCACT GCGTAAGCTA 1200 ATTGATATTC AATCGAATAC CGGTTGGACA AGTGGCGGTC ATACCGCTAT GGATGTCCAA 1260 GTCTTTGCTG AAGGCCCTGG CGCTAGATTA TTTAGCGGTC ATCAAGACAA TATTGATATT 1320 GCGATTAAAA TGTTTAGCTT ATTGCCACAA TCGGTGCAAA CACCTTAA 1368 <210> 3 <211> 30 <212> Nucleic acid <213> Artificial sequence <223> Synthetic primer for PCR amplification <400> 3 ACGGGGGCTA CCATGGATGA AGTCATTATG 30 <210> 4 <211> 24 <212> Nucleic acid <213> Artificial sequence <223> Synthetic primer for PCR amplification <400> 4 AAGGGTCGAC GTTGATAGCG GAGG 24

[Brief description of the drawings]

FIG. 1 shows the gene sequence and amino acid sequence of SIB1-derived APase.

FIG. 2 is a continuation of the sequence shown in FIG.
It is a figure which shows the gene sequence and amino acid sequence of origin APase.

FIG. 3 is a diagram showing the construction of an Apase expression vector pET-AP5.

FIG. 4 is a schematic diagram showing a synthetic oligonucleotide for constructing an expression vector.

FIG. 5 is a graph showing the temperature dependence of APase activity.

FIG. 6 is a graph showing the pH dependence of APase activity.

FIG. 7 is a graph showing the thermal stability of APase.

Continued on the front page (72) Inventor Masaaki Morikawa 1-10-16 Onohara Nishi, Minoh-shi, Osaka (72) Inventor Mitsuru Haruki 2-1 Takemidai, Suita-shi, Osaka C7-403 F-term (reference) 4B024 AA20 BA11 CA01 HA08 4B050 CC01 DD02 EE10 LL05 LL10 4B064 AA01 CA21 CB04 CC24 CD15 DA20

Claims (5)

[Claims] 1. Shewanella sp. SIB having the following properties:
A protein derived from one strain: (1) catalyzes a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions to produce inorganic phosphate;
(2) molecular weight is 49 kDa; (3) isoelectric point is 4.9; (4) optimal temperature of alkaline phosphatase activity is in the range of 30 ° C. to 40 ° C .; 5) At 70 ° C, the alkaline phosphatase activity disappears within 10 minutes. 2. Sh having alkaline phosphatase activity
A polypeptide derived from ewanella sp. SIB1 strain, which comprises an amino acid sequence represented by the following (a) or (b): (A) amino acid numbers 1-455 shown in SEQ ID NO: 1 in the sequence listing
A polypeptide comprising an amino acid sequence represented by the formula: (B) producing an inorganic phosphoric acid by catalyzing a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions;
A polypeptide having an alkaline phosphatase activity, wherein a part of (a) is deleted, substituted or added. 3. A gene encoding the polypeptide according to claim 2. 4. Sh having alkaline phosphatase activity
ewanella sp. encodes a polypeptide derived from SIB1 strain,
A gene comprising a base sequence shown in the following (c) or (d): (C) a gene comprising a nucleotide sequence represented by nucleotide numbers 1-1368 shown in SEQ ID NO: 2 of the sequence listing. (D) generating an inorganic phosphoric acid by catalyzing a hydrolysis reaction using a phosphate monoester as a substrate under alkaline conditions;
A gene encoding a polypeptide having an alkaline phosphatase activity, wherein part (c) is deleted, substituted or added. 5. A method for hydrolyzing a phosphate monoester in DNA or a fragment thereof or RNA or a fragment thereof using the enzyme according to claim 1.