KR100861585B1 - Thermostable Pyrophosphatase from Picrophilus torridus - Google Patents

Abstract

The present invention relates to a heat-resistant pyrophosphatase derived from Picrophilus torridus, a gene encoding the same, and a recombinant vector including the same. It relates to a heat-resistant pyrophosphatase having an amino acid sequence set forth in SEQ ID NO: 2, a gene encoding the same, a recombinant vector comprising the gene, and a transformant transformed with the recombinant vector. Pyrophyllus toridus-derived pyrophosphatase of the present invention is a hot start PCR reaction mixture and DNA sequencing for inhibiting nonspecific amplification and primer-dimer formation that occur during PCR reactions. It can be usefully used for molecular biological methods such as enhancing the efficiency of the).

Pyrophosphatase, Pyrophyllus torridus, Pyrophosphate

Description

Heat-resistant pyrophosphatase derived from Pyrophyllus torsidus {Thermostable Pyrophosphatase from Picrophilus torridus}

FIG. 1 compares the amino acid sequence of Pyrophilus torridus-derived pyrophosphatase (hereinafter abbreviated as "PPase") with that of other species-derived PPases.

Pto; Pyrophyllus tordus derived PPase (SEQ ID NO: 2),

Tae; Thermoplasma ashdophylium-derived PPase (SEQ ID NO: 4),

Mth; PPase (SEQ ID NO: 5) derived from Metanopyrus candelli,

Pho; Pyrococcus horikoshi derived PPase (SEQ ID NO: 6),

Tli; Thermococcus littoralis-derived PPase (SEQ ID NO: 7),

Tth; PPase from Thermos Thermophilus (SEQ ID NO: 8),

*; Amino acid sequence conservation site

Figure 2 shows the ORF (open reading frame) sequence (SEQ ID NO: 3) of the Pyclophilus toridus-derived PPase gene having EcoRI and XhoI restriction enzyme recognition sites at the 5'-end and 3'-end, respectively.

Figure 3 is the result of electrophoresis of the Pyclophilus toridus derived PPase gene DNA obtained through gene synthesis.

FIG. 4 is a schematic diagram of the recombinant plasmid pPROP for producing Pyclophilus torridus-derived PPase in Escherichia coli by ligation of the Pyclophilus torridus-derived PPase gene into an expression vector.

Figure 5 is a photograph showing the results of the SDS-PAGE electrophoresis step of purification of Pyclophilus toridus-derived PPase.

Lane 1; Protein molecular weight markers,

Lane 2; A sample in which E. coli was not pulverized by a sample that did not express the Pto PPase gene,

Lane 3; A sample in which the sample expressing the Pto PPase gene in Escherichia coli was disrupted by high frequency sonication,

Lane 4; A sample of lane 3 was heat-treated at 75 ° C. for 30 minutes to remove E. coli-derived protein by centrifugation,

Lane 5; Sample of lane 4 purified by Ni-NTA column chromatography

Figure 6 is a graph showing the relative activity according to the temperature of the purified Pyrophyllus toridus-derived PPase.

7 is a graph showing the relative activity according to the pH of the purified Pyrophyllus toridus-derived PPase.

●; Citrate buffer, Δ; Tris-HCl buffer, ■; Glycine-NaOH Buffer

FIG. 8 is a graph showing the relative activity of the divalent cations of purified Pyrophyllus toryose derived PPase.

The present invention relates to a heat-resistant PPase and a decomposition method of pyrophosphate using the same.

PPase catalyzes the reaction of pyrophosphate to hydrolyze into phosphates and is an enzyme that plays a very important role in the energy metabolism of cells (Lahti, R., Microbiol. Rev. 47: 169-179, 1983). PPase is a divalent cation, such as Mg ^{2 +} is needed to indicate a bacterial, yeast, widely distributed and active like plant. The active site structure or catalytic mechanism of PPase is well preserved in many species, and catalysis proceeds without the production of phosphorylated enzymes.

Heat-resistant PPase, which maintains its activity even at high temperatures, is used in hot start PCR reaction mixtures to suppress non-specific amplification and primer-dimer formation that occur during polymerase chain reaction (PCR) reactions. It can be applied to DNA sequencing to determine sequencing, and can be used together with DNA polymerase when amplifying DNA by PCR to easily remove pyrophosphate generated by DNA polymerization reaction to increase reaction efficiency. have. In fact, hot-start PCR reaction mixtures containing pyrophosphate and PPase can inhibit the production of non-specific amplification products, which can effectively produce only specific amplification products, thereby amplifying small copies of DNA, There is a report that it can be usefully used for the amplification used, or for suppressing non-specific amplification products generated during multiple PCR reactions (Korean Patent Application No. 1999-0004361). Studies on these heat-resistant PPases have been mainly carried out in strains of the genus Thermoplasma, Thermos, and Thermococcus.

However, the conventional PPases are mostly enzymes derived from mesophilic bacteria, so they have a disadvantage in that activity is easily lost during hot start PCR or DNA sequencing. Therefore, the development of a new heat-resistant PPase that is stable at high temperatures is required, but research on PPase derived from heat-resistant microorganisms is insignificant compared to the study of PPase produced from mesophilic bacteria.

On the other hand, Pyclophilus toridus is a type of heat-resistant acidophilic microorganism (thermoacidophile), which is known to have a gene that is assumed to be PPase as the entire genome sequencing is completed (Fuetterer, O., et al. , Proc. Natl. Acad. Sci. USA 101, 9091-9096, 2004; GenBank accession numbers AE017261), yet no enzymatic properties have been reported. Accordingly, the present inventors have tried to develop a PPase having a higher heat resistance than the conventional PPase, and as a result, it was confirmed that the PPase derived from Pyclophilus toridus has a higher heat resistance than the PPase derived from other conventional heat resistant strains, The present invention has been completed by revealing that the enzyme can be applied to various molecular biological experiments.

An object of the present invention is to provide a PPase, a gene encoding the enzyme, a recombinant vector to which the gene is fused, and a transformant transformed from the pyrophyllus torridus and having a stable activity at a high temperature. It is.

The present invention provides a PPase derived from Pyclophilus torridus and having stable activity at high temperatures.

The heat resistant PPase of the present invention comprises a gene sequence encoding a heat resistant PPase derived from a microorganism, preferably thermococcus littoralis (PCT Application No. PCT / US95 / 13662), in which the enzymatic properties of the heat resistant PPase are known. Based on the amino acid sequence inferred from the sequence, it can be secured by comparing and retrieving sequences presumed to be heat-resistant PPase in sequences from various heat-resistant strains. According to a preferred embodiment of the present invention, a thermophilic PPase derived from Pyclophilus torridus and a PPase having an amino acid sequence homology of 48.8% as a specific sequence was determined (see FIG. 1). The PPase has an amino acid sequence set forth in SEQ ID NO: 2 consisting of 177 amino acids.

According to a preferred embodiment of the present invention, the PPase derived from the Pyclophilus torridus has a molecular weight of about 23,900 Da (see Fig. 5), exhibits enzymatic activity in the temperature range of 65 ℃ to 85 ℃, at 80 ℃ Show the most optimal activity (see FIG. 6). In addition, it shows activity in a wide range of pH 7.0 to 9.0, and shows optimal activity at pH 7.5 (see FIG. 7). In addition, divalent cations are necessary to exhibit optimal activity, of which magnesium exhibits the most optimal activity (see FIG. 8).

The present invention also provides a gene encoding a PPase derived from the Pyclophilus torridus.

The gene of the present invention includes all nucleotide sequences capable of encoding a PPase derived from Pyrophyllus torridus as described in SEQ ID NO: 2, and preferably has a nucleotide sequence shown in SEQ ID NO: 1. The gene may comprise restriction enzyme recognition sites specific for its 5'- and / or 3'-ends to facilitate subsequent cloning and transformation. According to a preferred embodiment of the present invention, a gene having a length of 549 bp having a nucleotide sequence described in SEQ ID NO: 3 having restriction enzymes EcoRI and XhoI sites at the 5'-end and 3'-end of the gene, respectively, was synthesized. Conventional molecular biology methods can be used to prepare the gene without limitation, and in a preferred embodiment of the present invention, the gene was synthesized by a ligase chain reaction method (see FIGS. 2 and 3).

The present invention also provides a recombinant vector comprising the gene.

The recombinant vector of the present invention can be prepared by inserting a gene encoding the peptide described in SEQ ID NO: 2 in a general expression vector, preferably E. coli strain expression vector. In a preferred embodiment of the present invention, pET22b is used as an E. coli expression vector, but is not necessarily limited thereto, and all kinds of expression vectors that can be generally used may be used without limitation. In a preferred embodiment of the present invention, a recombinant vector was prepared by inserting a DNA fragment containing a gene encoding a PPase derived from Pyrophyllus tordus as set forth in SEQ ID NO: 3 using the pET22b vector, which is an E. coli strain expression vector. This was named "pPROP" (see Figure 4).

The present invention also provides a transformant transformed with the recombinant vector.

The transformant can be readily prepared by introducing the preparation vector into any host cell, preferably E. coli cells. In a preferred embodiment of the present invention, a transformant having a pPROP vector introduced into E. coli strains DH5α and BL21 (DE3) was prepared. The inventors named the recombinant Escherichia coli strain having high PPase activity as "BL21 (DE3) / pPROP", and deposited it on October 18, 2006 to the Korea Institute of Bioscience and Biotechnology Resource Center (Accession Number: KCTC 11003BP).

In addition, the present invention provides a method for producing PPase derived from Pyrophyllus toryose using the transformed cells.

PPase derived from the Pyclophilus toridus can be easily expressed and mass-produced by culturing the transformed cells in an appropriate medium, or introducing the transformed cells into any animal and then culturing in vivo. Can be. In a preferred embodiment of the present invention, the recombinant expression vector pPROP, in which the PPase gene derived from Pyrophyllus tordus is inserted into the pET22b vector, is introduced into Escherichia coli strain BL21 (DE3) to make a transformant, and the resulting peak is cultured in an appropriate medium. PPase derived from Rophyllus torytus was expressed and purified (see FIG. 5).

Pyclophilus torridus-derived PPase of the present invention can stably maintain activity even at a high temperature, preferably at a temperature of 80 ° C. or higher, and thus is used for inhibiting non-specific amplification products in various molecular biological experiments, such as a hot start PCR reaction. It can be usefully used. In addition, general molecular biological experimental methods related to the genetic manipulation, transformation, etc. are known methods, for example, "molecular cloning" of Sambrook et al. (Sambrook, J. et al. (1989) Molecular Cloning. A laboratory Manual. Cold Spring Harbor Laboratory.Cold Spring Harbor.New York), Osbel et al., "Simple Protocols in Molecular Biology" (Ausubel, F. et al. (1995) Short Protocols in Molecular Biology.3rd.John & Wiley Sons, Inc.). It can be performed easily according to the method such as).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for explaining the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by the following examples. .

Example 1 Selection of New Heat-Resistant PPase Gene

The US National Biotechnology Information Center homepage (http: // www) is based on the gene sequence encoding the PPase derived from Thermococcus litoralis and the amino acid sequence deduced from the sequence. blast search, an Internet program of .ncbi.nlm.nih.gov /), was used to obtain sequences presumed to be PPase in the entire genome sequence from various strains. Among them, genes having a sequence different from that of PPase, which had already been identified as an enzyme having high heat resistance, were selected.

As a result, the Pyclophyllus toricose-derived PPase having 0% gene sequence homology and 48.8% amino acid sequence homology with the thermococcus littoralis-derived PPase was determined as a specific sequence. The putative gene has a nucleotide sequence as set out in SEQ ID NO: 1 with a total of 534 bp including the stop codon, and encodes a protein consisting of 177 amino acids set forth in SEQ ID NO: 2. In addition, as shown in FIG. 1, as a result of comparing the amino acid sequences, the Pyclophilus torridus-derived PPase is 53% of the thermoplasma axidophylium-derived PPase belonging to the high temperature bacteria and 37% of the amino acid thermophilus-derived PPase Have the same sex.

<Example 2> Isolation of Pyclophilus torilose-derived PPase gene

549 bp in length having the nucleotide sequence described in SEQ ID NO: 3 having the restriction enzymes EcoRI and XhoI sites at the 5'- and 3'-terminals of the Pyclophilus torridus-derived PPase gene, as confirmed in Example 1, respectively. In order to synthesize the gene, the LCR method of Lauricard and Lee (Rouillard, JM, and Lee, W., Nucleic Acids Research, 2004, Vol. 32, 176-180) was modified as follows. The gene described in SEQ ID NO: 3 is prepared by adding an EcoRI recognition site to the 5 'end of SEQ ID NO: 1, removing the tga base sequence which is a stop codon present at the 3' end, and then adding the XhoI recognition site. At this time, the tga stop codon removed above will be used in the vector. The LCR method is a technology capable of synthesizing a gene without a template gene, and thus, efficient expression may be obtained through codon optimization.

To this end, in order to prepare the nucleotide sequence described in SEQ ID NO: 3 of the target gene, the Internet program Jin 2 oligo (http://berry.engin.umich.edu/gene2oligo/, Rouillard, JM, and Lee, W. , Nucleic Acids Res., 2004, Vol. 32, 176-180), designed 41 oligomers modified with 5'-phosphate at the 5'-end so that the Tm value was the same at 57 ° C. 2, SEQ ID NO: 9 to SEQ ID NO: 49). Dilute the oligomers by 5 pmole each and use Tfi ligase (ligase, manufactured by Bioneer, Korea) for 2 minutes at 94 ° C, 30 seconds at 94 ° C, and 4 minutes (40 cycles) at 57 ° C. LCR was performed under conditions.

Next, PCR was performed using the LCR product as template DNA and the oligomers set forth in SEQ ID NO: 9 and SEQ ID NO: 49 as the front and back primers, respectively. Experimental method was denatured template DNA for 5 minutes at 94 ℃, and then repeated 30 times in the order of 40 seconds at 94 ℃, 40 seconds at 57 ℃, 40 seconds at 72 ℃ by adding 2.5 units of Tag polymerase 72 The polymerization was carried out for a long time at 5 ° C. After completion of the reaction by electrophoresis on a 0.6% agarose (agarose) gel to extract the portion corresponding to the DNA of the desired size on the developed gel (Fig. 3), gel purification kit (gel purification kit, bioneer, Korea) Pyrophyllus toryose-derived PPase gene was purified using.

<Example 3> Cloning of Pyclophilus torilose PPase Gene

The target DNA fragment purified in Example 2 was digested with EcoRI and XhoI restriction enzymes. The cleaved DNA fragment was ligated with plasmid pET22b (Novagen, Germany) digested with EcoRI and XhoI to prepare plasmid "pPROP" (5.9 kb). Plasmid pPROP, in turn, consists of a T7-Lac fusion promoter, a Pyclophilus toricose-derived PPase structural gene, and a T7 transcription terminator. 4 shows a staining map of the recombinant expression vector pPROP.

Example 4 Preparation of Transformant

The plasmid pPROP prepared in Example 3 was transformed into Escherichia coli DH5α (recA1 endA1 gyrA96 thi1 hsdR17 supE44 relA1 lacZ M15, manufactured by invitrogen, USA) according to the CaCl ₂ method. To this end, first, E. coli DH5α was inoculated in 3 ml of LB medium (0.5% yeast extract, 1% bacto-tryptone, 0.5% sodium chloride) and shaken at 37 ° C for 12 hours. 200 μl of the culture was inoculated again in 20 ml of fresh LB medium, followed by shaking culture at 37 ° C. for 2-3 hours until the absorbance (OD ₆₀₀ ) of the culture solution became 600 at 600 nm. Thereafter, 20 ml of the culture solution was centrifuged at 4 ° C. at 8,000 rpm for 10 minutes to obtain a cell precipitate, and then, 5 ml of a 50 mM CaCl ₂ solution pre-cooled at 4 ° C. was added thereto and left at 4 ° C. for 10 minutes. It was. Separating 10 minutes and centrifuged at 8,000 rpm in 4 ℃ After removal of the CaCl ₂ solution, the CaCl ₂ solution put back 5 ㎖ was removed once more CaCl ₂ solution in the same manner as described above. 2 ml of 50 mM CaCl _{2 and} 1 ml of 45% glycerol solution were added thereto, and left at 4 ° C. for at least 30 minutes, and 100 µl of this was dispensed into an Eppendorf tube and used as a strain for transformation. The transformation strain was stored and used at -70 ℃.

The plasmid pPROP prepared in Example 3 was put into an Eppendorf tube containing a strain for transformation, and the first transformed to E. coli DH5α. The amplified plasmid pPROP was subjected to secondary transformation by Escherichia coli BL21 (DE3), a strain capable of stably mass-expressing Pyclophilus torydus-derived PPase, by the same CaCl ₂ method.

Example 5 Selection of Transformed Strains

In order to select strains having high PPase activity among the strains transformed in Example 4, the strains were inoculated in 3 ml LBA medium (LB + ampicillin 50 μl / ml), respectively, and then pre-incubated, and the strain culture solution was The test tube containing fresh 10 ml of LBA medium was inoculated at a concentration of 5%. When the absorbance (OD ₆₀₀ ) of the culture medium is 0.5, that is, when the strain is in the exponential phase, 0.5 mM IPTG (isopropyl-D-thio-galactopyranoside) is added, and the growth of the strain for each strain (cell recovery) ) And PPase activity were measured.

The recovered cells were suspended in a buffer solution (50 mM Tris-HCl, pH 8.0 / 5 mM MgCl ₂ ), and then the cells were disrupted by using an ultrasonicator. The crushed cells were centrifuged (13,000 rpm, 20 minutes) to recover the supernatant, and heat-treated at 75 ° C. for 30 minutes to remove E. coli-derived proteins. The heat-treated sample was centrifuged (13,000 rpm, 20 minutes), and the supernatant recovered was used as a crude enzyme solution.

400 μl of a pyrophosphate solution as a substrate was added to 100 μl of the coenzyme solution (final substrate concentration: 0.32 mM) for 10 minutes at 65 ° C., and then a phosphate reaction solution (1% NH ₄ ) ₆ Mo ₇ O ₂₄ 4H ₂ O (w / v), 8% FeSO ₄ · 7H ₂ O (w / v), 0.83 MH ₂ SO ₄ ) was added 1 ml and mixed well, at 660 nm The absorbance was measured to select strains with high activity. Growth of the strain was measured by comparing the absorbance (OD ₆₀₀ ) based on the culture. At this time, the PPase enzyme activity was expressed in units (unit / ㎖), 1 unit was defined as the amount of enzyme to produce 40 nmole of phosphate from pyrophosphate for 1 minute at 65 ℃.

The present inventors named the recombinant Escherichia coli strain having high PPase activity selected above as "BL21 (DE3) / pPROP" and deposited it at the Korea Research Institute of Bioscience and Biotechnology, October 18, 2006 (Accession No .: KCTC 11003BP). ).

Example 6 Optimal Expression and Purification of Pyclophilus torridus-derived PPase Gene

The recombinant strain BL21 (DE3) / pPROP selected in Example 5 was inoculated into 20 ml of LBA medium (ampicillin, 50 μg / ml), preincubated at 37 ° C. and 180 rpm for about 9 hours, and then 500 ml of LBA medium. (ampicillin, 50 ㎍ / ㎖) was inoculated 1, 3, 5% respectively to perform the main culture. During the main culture, when the absorbance (OD ₆₀₀ ) of the culture solution was 0.2, 0.6, and 0.8, respectively, the PPase gene was expressed while culturing with time by adding IPTG with final concentrations of 0.1, 0.5, and 1.0 mM. As a result, when the 1% inoculation of the culture, the absorbance of the culture was 0.6, the most optimal expression was obtained when IPTG of 0.1 mM final concentration was added.

In order to purify the PPase, the cells were suspended in a buffer containing 1 mM PMSF (50 mM Tris-HCl, pH 8.0 / 5 mM MgCl ₂ ), the cells were disrupted by amplification and centrifuged to recover the supernatant. The recovered supernatant was heat-treated at 75 ° C. for 30 minutes, and then centrifuged again to remove E. coli-derived protein. The supernatant was loaded onto a Ni-NTA column equilibrated with lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM C ₃ H ₄ N ₂ (imidazole), pH 8.0), followed by washing ( washing unbound protein with washing buffer; 50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 mM imidazole, pH 8.0). Then, the bound Pyrophyllus toryose-derived PPase was isolated and purified while gradually changing the imidazole concentration to 50-300 mM. After confirming the fractions eluted by the method of measuring the PPase activity of Example 5 and dialyzed for about 24 hours, SDS-PAGE electrophoresis was performed.

As a result, as shown in FIG. 5, the Pyclophilus torilus-derived PPase was found to have a subunit having a molecular weight of about 23,900 Da.

<Example 7> Characterization of Pyclophilus torridus-derived PPase

<7-1> optimum active temperature

The properties of the enzyme were examined using the Pyclophilus torridus-derived PPase purified in Example 6, wherein the activity of the enzyme was measured according to the method of Example 5. In order to determine the optimal activity temperature of the purified Pyclophilus torridus-derived PPase, the temperature range was investigated at intervals of 5 to 10 ° C. from 40 ° C. to 99 ° C.

As a result, as shown in Figure 6, it showed a variety of enzyme activity from 65 ℃ to 85 ℃, of which showed the most optimal activity at 80 ℃.

<7-2> optimum active pH

In order to determine the optimal active pH of the Pyclophyllus todoridus-derived PPase, a buffer of each pH was mixed at 100 μl of PPase purified at an interval of 0.4 to 1.0 at a pH of 3.0 to 11.0 at a concentration of 50 mM, followed by The pyrophosphate solution was added and reacted at 65 ° C. for 10 minutes to measure enzymatic activity by the PPase activity measurement method described in Example 5. In this case, pH 3.0-6.0 was used as 50 mM citrate buffer, pH 6.0-8.0 was used as 50 mM Tris-HCl buffer, and pH 8.0-11.0 was used as glycine-NaOH buffer.

As a result, as shown in Fig. 7, Pyclophilus torridus-derived PPase showed high activity in Tris-HCl buffer solution, and showed activity in a wide range ranging from pH 7.0 to 9.0, among which the optimum at pH 7.5 Activity was shown.

<7-3> Measurement of the Effect of Divalent Cations

In order to investigate the effects of divalent cations (MgCl ₂ , ZnCl ₂ , CoCl ₂ , MnCl ₂ , CuCl ₂ , DTT, CaCl ₂ , EDTA) on the activity of Pyclophilus torsidose-derived PPase, the following experiments were carried out: It was. After dilution cation was added to 100 μl of purified Pyrophyllus toridus-derived PPase enzyme solution and 50 mM Tris-HCl buffer (pH 7.5) such that the final concentration was 1-5 mM, and the mixture was left at room temperature for 1 hour. Pyrophosphate solution as a substrate was added to each sample and reacted at 65 ° C for 10 minutes. Thereafter, PPase activity was measured in the same manner as in Example 5.

As a result, as shown in FIG. 8, when MgCl ₂ , ZnCl ₂ , CoCl ₂ , MnCl ₂ and CuCl ₂ were added, the activity was higher than that of the control group without enzymatic reaction without divalent cation. The highest activity was obtained when MgCl ₂ was used.

As described above, the pyrophyllus torridus-derived heat-resistant PPase of the present invention can stably exhibit activity at a higher temperature than the conventional heat-resistant PPase, thereby inhibiting non-specific amplification and primer-dimer formation generated during PCR reaction. It can be used for hot start PCR reaction mixtures, and can be usefully used to improve the efficiency of sequencing reacting at high temperature.

<110> Bioneer Corporation <120> Thermostable Pyrophosphatase from Picrophilus torridus <130> 2006DPA120 <160> 49 <170> KopatentIn 1.71 <210> 1 <211> 534 <212> DNA <213> Picrophilus torridus <400> 1 atggaaaaga atatgagcta ctggcaccag gtacctccag gcccaaaccc gcctgatgag 60 gtctatgttg tcgtggagat accaaaaggt gagaggaaca aatatgagat agcaaaggag 120 tttcccggca taaagcttga tagaataata tactcatcat atgtttatcc tcttgagtac 180 ggtttgatac cgcagaccta ttactctgat ggggatccaa tagatgcaat ggtctttatg 240 tcacagagca cctatccggg cgttatatta agggcaaaac cggtcggcat gatgaacatg 300 gttgattccg gggacgtcga taataaaatt atatgtgtgt gccttgatga ccctgtttat 360 tccaagataa ataactacag ggaaatacct gagcatgtat taaaggaaac cgagaacttc 420 tttgaaacat acaaaaaact gcaaaataaa gaggtaaagg ttaccggctg ggaaggccct 480 gataaggcca agcaggagat aaaaaaggca atagaagatt ataaaaaact atga 534 <210> 2 <211> 177 <212> PRT <213> Picrophilus torridus <400> 2 Met Glu Lys Asn Met Ser Tyr Trp His Gln Val Pro Pro Gly Pro Asn   1 5 10 15 Pro Pro Asp Glu Val Tyr Val Val Val Glu Ile Pro Lys Gly Glu Arg              20 25 30 Asn Lys Tyr Glu Ile Ala Lys Glu Phe Pro Gly Ile Lys Leu Asp Arg          35 40 45 Ile Ile Tyr Ser Ser Tyr Val Tyr Pro Leu Glu Tyr Gly Leu Ile Pro      50 55 60 Gln Thr Tyr Tyr Ser Asp Gly Asp Pro Ile Asp Ala Met Val Phe Met  65 70 75 80 Ser Gln Ser Thr Tyr Pro Gly Val Ile Leu Arg Ala Lys Pro Val Gly                  85 90 95 Met Met Asn Met Val Asp Ser Gly Asp Val Asp Asn Lys Ile Ile Cys             100 105 110 Val Cys Leu Asp Asp Pro Val Tyr Ser Lys Ile Asn Asn Tyr Arg Glu         115 120 125 Ile Pro Glu His Val Leu Lys Glu Thr Glu Asn Phe Phe Glu Thr Tyr     130 135 140 Lys Lys Leu Gln Asn Lys Glu Val Lys Val Thr Gly Trp Glu Gly Pro 145 150 155 160 Asp Lys Ala Lys Gln Glu Ile Lys Lys Ala Ile Glu Asp Tyr Lys Lys                 165 170 175 Leu     <210> 3 <211> 549 <212> DNA <213> Picrophilus torridus <220> <221> misc_feature (222) (4) .. (9) <223> EcoRI recognition site <220> <221> misc_feature <542> (542) .. (547) <223> XhoI recognition site <400> 3 ccggaattcg atggaaaaga atatgagcta ctggcaccag gtacctccag gcccaaaccc 60 gcctgatgag gtctatgttg tcgtggagat accaaaaggt gagaggaaca aatatgagat 120 agcaaaggag tttcccggca taaagcttga tagaataata tactcatcat atgtttatcc 180 tcttgagtac ggtttgatac cgcagaccta ttactctgat ggggatccaa tagatgcaat 240 ggtctttatg tcacagagca cctatccggg cgttatatta agggcaaaac cggtcggcat 300 gatgaacatg gttgattccg gggacgtcga taataaaatt atatgtgtgt gccttgatga 360 ccctgtttat tccaagataa ataactacag ggaaatacct gagcatgtat taaaggaaac 420 cgagaacttc tttgaaacat acaaaaaact gcaaaataaa gaggtaaagg ttaccggctg 480 ggaaggccct gataaggcca agcaggagat aaaaaaggca atagaagatt ataaaaaact 540 actcgagcg 549 <210> 4 <211> 540 <212> DNA <213> Thermoplasma acidophilum DSM 1728 <400> 4 atggagagct tttatcattc ggttccagtt ggcccaaagc cgccagagga ggtctacgtc 60 atcgtagaga tcccaagggg aagcagggtg aagtacgaga tagccaagga ctttccgggc 120 atgctagttg acagggtgct ctattcatcc gttgtttatc ctgttgatta cggtctcatt 180 cccagaacgc tttattatga tggagatccc atggacgtga tggttctcat atcgcagccg 240 actttccccg gagccatcat gaaggtcagg cccatcggca tgatgaagat ggtggatcag 300 ggtgaaacgg acaataagat actcgcagtt ttcgacaagg atcccaatgt cagctacata 360 aaggatctga aggatgtcaa tgcccatctg cttgatgaga tagcaaactt cttctcaacg 420 tacaagatcc tggagaagaa ggagacaaag gttcttggat gggagggcaa ggaggctgcc 480 ctaaaggaga tagaagtgtc tattaagatg tatgaggaga aatacggaaa gaagaactag 540                                                                          540 <210> 5 <211> 531 <212> DNA <213> Methanopyrus kandleri AV19 <400> 5 atgatgaacc tctggaaaga cctggaaccg ggcccgaatc cacccgacgt agtgtacgcg 60 gtgatagaaa tcccacgtgg ctcgaggaac aagtacgagt acgacgaaga gcgtgggttc 120 ttcaagctgg accgagtact ctactcgccg ttccactatc cactggacta cgggtttatc 180 ccccgaacgc tgtacgacga cggtgacccc ctcgacatcc tggtgatcat gcaggacccc 240 acgttccccg gctgcgtgat cgaggcacga ccgatcggtc tgatgaagat gttggacgac 300 agcgaccagg acgacaaggt tctggcggtg cccactgagg acccccggtt caaggacgtt 360 aaggacctgg atgacgttcc caagcacctg ctcgacgaga tcgcccacat gttctccgag 420 tacaagcgac tggaaggcaa ggagaccgaa gtcctagggt gggaaggtgc ggacgccgct 480 aaggaagcca tagttcacgc gatagagctg tacgaggagg agcacgggtg a 531 <210> 6 <211> 537 <212> DNA <213> Pyrococcus horikoshii OT3 <400> 6 atgaacccat tccacgacct tgaacccgga ccaaacgttc cagaagttgt ttacgccctg 60 atagagatac cgaagggaag tagaaacaag tacgagctag ataaggaaac gggattgttg 120 aaattagata gggttctcta cacaccgttt cactacccag tagactacgg gataatacca 180 aggacatggt atgaagatgg agatcccttt gacatcatgg taattatgag ggaaccaacg 240 tatccactaa cgataataga ggccaggccg atcggactat ttaaaatgat agacagcgga 300 gataaggatt ataaggtact tgcagttcca gtcgaagatc catactttaa agactggaag 360 gatataagcg atgttcccaa ggcattctta gacgagatag cccacttctt caagaggtat 420 aaggaactcg aaggtaaaga gataatcgta gaaggatggg aaggggcaga agctgctaag 480 agggaaattt taagggctat agagatgtat aaggagaaat tcggcaagaa ggagtga 537 <210> 7 <211> 531 <212> DNA <213> Thermococcus litoralis <400> 7 atgaatccat tccacgattt agagcctgga ccggaagtac cggaagttgt ttacgcctta 60 atagagattc caaaggggag cagaaacaag tatgagcttg acaaaaagac cggccttata 120 aagctcgata gagttcttta cagcccattc cactacccgg tcgactatgg aatcatccca 180 caaacatggt acgatgatga cgacccgttt gacatcatgg tcataatgag ggagccaaca 240 tatccgggag ttcttattga ggcaagacca ataggcctct tcaagatgat agacagcggc 300 gacaaggact acaaggtatt ggcagttcca gtggaagatc cctactttaa tgactggaag 360 gacataagcg acgttccgaa ggctttcctt gacgagattg cgcacttctt ccagagatac 420 aaagagctcc aaggtaagga aatcattgtt gagggctggg aaaacgcaga gaaggcaaag 480 caagaaatac ttagggcaat agaactttac aaggagaaat tcaagaagtg a 531 <210> 8 <211> 528 <212> DNA <213> Thermus thermophilus <400> 8 atggcgaacc tgaagagcct tcccgtgggc gacaaggcgc ccgaggtggt ccacatggtc 60 attgaggtcc cccgcggctc gggcaacaag tacgagtacg acccggacct cggggcgatc 120 aagctggacc gggtcctgcc gggagcccag ttctaccccg gggactacgg cttcatcccc 180 tccaccctgg ccgaggacgg ggaccccttg gacggcctcg tcctctccac ctaccccctc 240 ctccccgggg tggtggtgga ggtccgggtg gtgggcctcc tcctcatgga ggacgagaag 300 ggcggggatg ccaaggtcat cggggtggtg gccgaggacc agcgcctgga ccacatccag 360 gacatcgggg acgtccccga gggcgtgaag caagagatcc agcacttctt tgagacctac 420 aaggccctcg aggccaagaa ggggaagtgg gtcaaggtca cgggctggcg ggaccggaag 480 gcggccttgg aggaggtccg ggcctgcatc gcccgctaca agggctag 528 <210> 9 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> primer R1 <400> 9 catcgaattc cgg 13 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer F1 <400> 10 ccggaattcg atggaaaaga atatgagc 28 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer R2 <400> 11 tggtgccagt agctcatatt cttttc 26 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer F2 <400> 12 tactggcacc aggtacctcc agg 23 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer R3 <400> 13 gcgggtttgg gcctggaggt acc 23 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer F3 <400> 14 cccaaacccg cctgatgagg tctat 25 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer R4 <400> 15 ctccacgaca acatagacct catcag 26 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer F4 <400> 16 gttgtcgtgg agataccaaa aggtga 26 <210> 17 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer R5 <400> 17 tcatatttgt tcctctcacc ttttggtat 29 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer F5 <400> 18 gaggaacaaa tatgagatag caaaggagt 29 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer R6 <400> 19 tatgccggga aactcctttg ctatc 25 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer F6 <400> 20 ttcccggcat aaagcttgat agaataa 27 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer R7 <400> 21 aacatatgat gagtatatta ttctatcaag ctt 33 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer F7 <400> 22 tatactcatc atatgtttat cctcttgagt ac 32 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer R8 <400> 23 gcggtatcaa accgtactca agaggata 28 <210> 24 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer F8 <400> 24 ggtttgatac cgcagaccta ttactctg 28 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer R9 <400> 25 ttggatcccc atcagagtaa taggtct 27 <210> 26 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer F9 <400> 26 atggggatcc aatagatgca atggt 25 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer R10 <400> 27 ctctgtgaca taaagaccat tgcatcta 28 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer F10 <400> 28 ctttatgtca cagagcacct atccggg 27 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer R11 <400> 29 gcccttaata taacgcccgg ataggtg 27 <210> 30 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer F11 <400> 30 cgttatatta agggcaaaac cggtcg 26 <210> 31 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer R12 <400> 31 atgttcatca tgccgaccgg tttt 24 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer F12 <400> 32 gcatgatgaa catggttgat tccgg 25 <210> 33 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer R13 <400> 33 attatcgacg tccccggaat caacc 25 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer F13 <400> 34 ggacgtcgat aataaaatta tatgtgtgtg 30 <210> 35 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer R14 <400> 35 gggtcatcaa ggcacacaca tataatttt 29 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer F14 <400> 36 ccttgatgac cctgtttatt ccaagat 27 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer R15 <400> 37 tccctgtagt tatttatctt ggaataaaca 30 <210> 38 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer F15 <400> 38 aaataactac agggaaatac ctgagcat 28 <210> 39 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer R16 <400> 39 cggtttcctt taatacatgc tcaggtatt 29 <210> 40 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer F16 <400> 40 gtattaaagg aaaccgagaa cttctttgaa a 31 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer R17 <400> 41 gcagtttttt gtatgtttca aagaagttct 30 <210> 42 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer F17 <400> 42 catacaaaaa actgcaaaat aaagaggtaa ag 32 <210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer R18 <400> 43 cagccggtaa cctttacctc tttatttt 28 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer F18 <400> 44 gttaccggct gggaaggccc tga 23 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer R19 <400> 45 gcttggcctt atcagggcct tcc 23 <210> 46 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer F19 <400> 46 taaggccaag caggagataa aaaagg 26 <210> 47 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer R20 <400> 47 tttttataat cttctattgc cttttttatc tcct 34 <210> 48 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer F20 <400> 48 caatagaaga ttataaaaaa ctactcgagc g 31 <210> 49 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> primer R21 <400> 49 cgctcgagta gt 12  

Claims (12)

delete delete delete delete delete delete delete delete A transformant transformed with the recombinant expression vector pPROP comprising a pyrophosphatase gene having the nucleotide sequence set forth in SEQ ID NO: 1 is cultured in a medium, or in vivo after the transformant is introduced into any animal. Thereby producing a pyrophyllus toridus-derived pyrophosphatase having an amino acid sequence as set forth in SEQ ID NO: 2. A method of decomposing pyrophosphate into phosphates by applying pyrophyllus tordus derived pyrophosphatase having an amino acid sequence set forth in SEQ ID NO: 2 to pyrophosphate. The method of claim 10, Said decomposition being carried out in the presence of a divalent cation at a temperature of 65 ° C. to 85 ° C., at a pH range of 7.0 to 9.0. The method of claim 11, The decomposition is carried out at 80 ° C., pH 7.5 in the presence of MgCl ₂ .

Images