Method for producing nucleoside-5′-phosphate ester

Purification and Characterization of Acid Phosphatase Derived from Morganella morganii

A nutrient medium (pH 7.0, 50 ml) containing 1 g/dl of peptone, 0.5 g/dl of yeast extract, and 1 g/dl of sodium chloride was poured into Sakaguchi flasks (500 ml), which were sterilized at 120° C. for 20 minutes. A slant culture of Morganella morganii NCIMB 10466 was inoculated to each of the flasks once with a platinum loop, which was cultivated at 30° C. for 16 hours with shaking. Microbial cells (about 3,000 g), which were harvested from a culture by centrifugation, were suspended in 100 mM potassium phosphate buffer (1 L, pH 7.0). A ultrasonic treatment was performed at 4° C. for 20 minutes to disrupt the microbial cells. The treated suspension was centrifuged to remove the insoluble fraction. Thus, a cell-free extract was prepared.

Ammonium sulfate was added to the cell-free extract so that 30% saturation was achieved. Appeared precipitate was removed by centrifugation, and then ammonium sulfate was further added to supernatant so that 60% saturation was achieved. Appeared precipitate was collected by centrifugation, and it was dissolved in 100 mM potassium phosphate buffer.

This crude enzyme solution was dialyzed four times against 5 L of 100 mM potassium phosphate buffer (pH 7.0), and then it was applied to a DEAE-Toyopeal 650M column (φ4.1×22 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0), followed by washing with 800 of 20 mM potassium phosphate buffer (pH 7.0). The transphosphorylation activity was found in a fraction which passed through the column, and thus the fraction was recovered.

The fraction was added with ammonium sulfate so that 35% saturation was achieved, which was adsorbed to a Butyl-Toyopeal column (φ3.1×26 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing ammonium sulfate at 35% saturation. Elution was performed by using a linear concentration gradient from 35% saturation to 20% saturation in potassium phosphate buffer (pH 7.0).

Active fractions were collected and dialyzed against 1 L of 50 mM potassium phosphate buffer (pH 7.0), and then applied to a hydroxyapatite column (φ5×6.5 cm) equilibrated with 50 mM potassium phosphate buffer (pH 7.0). Elution was performed by using a linear concentration gradient from 50 mM to 300 mM of potassium phosphate buffer (pH 7.0).

Active fractions were collected and concentrated by ultrafiltration. This enzyme solution was applied into a HiLoad TM 16/60 Superdex 200 column (produced by Pharmacia). Elution was performed at a flow rate of 1.0 ml/minute by using 50 mM potassium phosphate buffer containing 100 mM sodium chloride.

In accordance with the procedure as described above, the enzyme exhibiting the transphosphorylation activity was purified from the cell-free extract consequently about 550-fold at a recovery ratio of about 10%. The specific activity and the recovery ratio in this purification process are shown in Table 1. This enzyme sample was homogeneous on SDS-polyacrylamide gel electrophoresis.

The purified enzyme had the following properties.

(1) Action: A Phosphate group was transferred from a phosphate group donor such as polyphosphoric acid, to a nucleoside, and nucleoside-5′-phosphate ester was produced. Reversely, this enzyme also exhibits an activity to hydrolyze phosphate ester.

(2) Substrate specificity: Phosphate group donors those which serve as the phosphate group donor in the transphosphorylation reaction include, for example, pyrophosphoric acid, tripolyphosphoric acid, trimetaphosphoric acid, tetrametaphosphoric acid, hexametaphosphoric acid, disodium phenylphosphate, dipotassium phenylphosphate, O,O-diphenylphosphoric acid anhydride, disodium carbamyl phosphate, dipotassium carbamyl phosphate, diammonium carbamyl phosphate, and dilithium carbamyl phosphate. Those which serve as the phosphate group acceptor include, for example, purine riboside, inosine, guanosine, adenosine, xanthosine, uridine, and cytidine. On the other hand, those which undergo the action in the phosphate ester hydrolytic reaction include, for example, inorganic phosphoric acid such as pyrophosphoric acid, tripolyphosphoric acid, trimetaphosphoric acid, tetrametaphosphoric acid, hexametaphosphoric acid; phosphate ester such as disodium phenylphosphate, dipotassium phenylphosphate, O,O-diphenylphosphoric acid anhydride, disodium carbamyl phosphate, dipotassium carbamyl phosphate, diammonium carbamyl phosphate, and dilithium carbamyl phosphate; and 5′-nucleotide such as 5′-purine ribotide, 5′-inosinic acid, 5′-guanylic acid, 5′-adenylic acid, 5′-xanthylic acid, 5′-uridylic acid, and 5′-cytidylic acid.

(3) Optimum pH: 5.2 (transphosphorylation reaction), 6.5 (phosphate ester hydrolytic reaction).

(4) pH stability: pH 3.0 to 12.0 (treatment at 30° C. for 60 minutes).

(5) Optimum temperature: about 35° C.

(6) Temperature stability: stable up to 30° C. (treatment at pH 7.0 for 30 minutes).

(7) Effect of the addition of metal ion and inhibitor: this enzyme exhibits no activation phenomenon relevant to its activity by addition of any metal ion. The activity is inhibited by Ag 2+ , Pb 2+ , Hg 2+ , and Cu 2+ . The activity is also inhibited by iodoacetic acid.

(8) Molecular weight: a calculated molecular weight is about 190,000 in accordance with high-performance liquid chromatography (TSKgel G-3000SW, produced by Toyo Soda).

(9) Sub unit molecular w eight: a calculated subunit molecular weight is about 25,000 in accordance with SDS-polyacrylamide gel electrophoresis.

This enzyme exhibits not only the activity to transfer phosphate group to a nucleoside, but also the activity to reversely hydrolyze a phosphate ester. Moreover, this enzyme exhibits the phosphate ester hydrolytic activity (phosphomonoesterase activity) which is higher than the transphosphorylation activity by not less than 20 times. Other properties are well coincident with those of a known acid phosphatase produced by a bacterium belonging to the genus Morganella ( Microbiology, 140, 1341-1350 (1994)). Accordingly, it has been clarified that this enzyme is an acid phosphatase.

Sodium pyrophosphate (10 g/dl) and inosine (2 g/dl) were dissolved in sodium acetate buffers each having pH of 5.5, 5.0, 4.5, 4.0, and 3.5, to which the enzyme sample described above was added so that a concentration of 50 units/dl was obtained. The reaction mixture was incubated at 30° C. for 6 hours while maintaining each pH, and the amount of produced 5′-inosinic acid was measured along with passage of time. Produced inosinic acid contained only 5′-inosinic acid. Production of 2′-inosinic acid and 3′-inosinic acid by-products was not observed at all. The result is shown in FIG. 1 . The velocity of 5′-inosinic acid production was maximum at pH 5.0. However, the maximum accumulated amount of 5′-inosinic acid was higher at lower pH. The reaction condition at pH 4.0 was most efficient for production of 5′-inosinic acid, in which 5′-inosinic acid was produced and accumulated in an amount of 2.60 g/dl by performing the reaction for 3 hours.

Phosphorylation Reaction of Various Nucleosides by Acid Phosphatase Sample Derived from Morganella morganii

Sodium pyrophosphate (10 g/dl) and inosine, guanosine, uridine, or cytidine (2 g/dl) as a phosphate group acceptor were dissolved in sodium acetate buffer (pH 4.0), to which the enzyme sample prepared in Example 1 was added so that its concentration was 50 units/dl. The reaction mixture was incubated at 30° C. for 3 hours while maintaining pH at 4.0. The amount of nucleoside-5′-ester produced by the reaction is shown in Table 2.

Produced nucleotide contained only nucleoside-5′-ester. Production of nucleoside-2′-ester and nucleoside-3′-ester by-products was not observed at all.

Production of 5′-Inosinic acid from Various Phosphate Compounds as Phosphate Group Donors by Acid Phosphatase Sample Derived from Morganella morganii

Inosine (2 g/dl) and sodium tripolyphosphate, sodium polyphosphate (trade name: Polygon P, produced by Chiyoda Chemical), disodium phenylphosphate, or disodium carbamyl phosphate (10 g/dl) as a phosphate group donor were dissolved in sodium acetate buffer (pH 4.0), to which the enzyme sample prepared in Example 1 was added so that its concentration was 50 units/dl. The reaction mixture was incubated at 30° C. for 3 hours while maintaining pH at 4.0. The amount of 5′-inosinic acid produced by the reaction is shown in Table 3.

5′-Inosinic acid was efficiently produced and accumulated by using any of the phosphate group donors. However, the accumulated amount of 5′-inosinic acid was the highest when sodium polyphosphate was used as the phosphate group donor.

Purification and Characterization of Acid Phosphatase Derived from Escherichia blattae

A nutrient medium (pH 7.0, 50 ml) containing 1 g/dl of peptone, 0.5 g/dl of yeast extract, and 1 g/dl of sodium chloride was poured into Sakaguchi flasks (500 ml), which was sterilized at 120° C. for 20 minutes. A slant culture of Escherichia blattae JCM 1650 was inoculated to each of the flasks once with a platinum loop, which was cultivated at 30° C. for 16 hours with shaking. Microbial cells were harvested from a culture by centrifugation. The microbial cells (about 3,300 g) were suspended in 100 mM potassium phosphate buffer (1 L, pH 7.0). A ultrasonic treatment was performed at 4° C. for 20 minutes to disrupt the microbial cells. The treated suspension was centrifuged to remove its insoluble fraction. Thus a cell-free extract was prepared.

Ammonium sulfate was added to the cell-free extract so that 30% saturation was achieved. Appeared precipitate was removed by centrifugation, and then ammonium sulfate was further added to supernatant so that 60% saturation was achieved. Appeared precipitate was recovered by centrifugation, and it was dissolved in 100 mM potassium phosphate buffer.

This crude enzyme solution was dialyzed four times against 5 L of 100 mM potassium phosphate buffer (pH 7.0), and then it was applied to a DEAE-Toyopeal 650M column (φ6.2×35 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0), followed by washing with 20 mM potassium phosphate buffer (pH 7.0). The transphosphorylation activity was found in a fraction which passed through the column, and thus the fraction was collected.

The active fraction was added with ammonium sulfate so that 35% saturation was achieved, which was applied to a Butyl-Toyopeal column (φ5.0×22.5 cm) equilibrated with 20 mM potassium phosphate buffer (pH 7.0) containing ammonium sulfate at 35% saturation. Elution was performed by using a linear concentration gradient from 35% saturation to 20% saturation in potassium phosphate buffer (pH 7.0).

Active fractions were collected and dialyzed against 1 L of 100 mM potassium phosphate buffer (pH 7.0), followed by being applied to a hydroxyapatite column (φ3.0×7.0 cm) equilibrated with 100 mM potassium phosphate buffer (pH 7.0). Elution was performed by using a linear concentration gradient from 50 mM to 100 mM of potassium phosphate buffer (pH 7.0), and active fractions were collected.

This enzyme solution was dialyzed against 1 L of 10 mM potassium phosphate buffer (pH 6.0), followed by being applied to a CM-Toyopearl column (φ2.0×14.0 cm) equilibrated with 10 mM potassium phosphate buffer (pH 6.0). Elution was performed by using a linear concentration gradient in potassium phosphate buffer (pH 6.0) containing from 0 mM to 300 mM potassium chloride. Active fractions eluted from the column were collected.

In accordance with the procedure as described above, the enzyme exhibiting the transphosphorylation activity was purified from the cell-free extract consequently about 600-fold at a recovery ratio of about 16% . The specific activity and the recovery ratio in this purification process are shown in Table 4. This enzyme sample was homogeneous on SDS-polyacrylamide gel electrophoresis.

The purified enzyme had the following properties.

(1) Action: A phosphate group was transferred from a phosphate group donor such as polyphosphoric acid to a nucleoside, and nucleoside-5′-phosphate ester was produced. Reversely, this enzyme also exhibits an activity to hydrolyze phosphate ester.

(2) Substrate specificity: Compounds which serve as the phosphate group donor in the transphosphorylation reaction include, for example, pyrophosphoric acid, tripolyphosphoric acid, trimetaphosphoric acid, tetrametaphosphoric acid, hexametaphosphoric acid, disodium phenylphosphate, dipotassium phenylphosphate, O,O-diphenylphosphoric acid anhydride, disodium carbamyl phosphate, dipotassium carbamyl phosphate, diammonium carbamyl phosphate, and dilithium carbamyl phosphate. Those which serve as the phosphate group acceptor include, for example, purine riboside, inosine, guanosine, adenosine, xanthosine, uridine, and cytidine. On the other hand, those which undergo the action in the phosphate ester hydrolytic reaction include, for example, inorganic phosphoric acid such as pyrophosphoric acid, tripolyphosphoric acid, trimetaphosphoric acid, tetrametaphosphoric acid, hexametaphosphoric acid; phosphate ester such as disodium phenylphosphate, dipotassium phenylphosphate, O,O-diphenylphosphoric acid anhydride, disodium carbamyl phosphate, dipotassium carbamyl phosphate, diammonium carbamyl phosphate, and dilithium carbamyl phosphate; and 5′-nucleotide such as 5′-purine ribotide, 5′-inosinic acid, 5′-guanylic acid, 5′-adenylic acid, 5′-xanthylic acid, 5′-uridylic acid, and 5′-cytidylic acid.

(3) Optimum pH: 5.2 (transphosphorylation reaction), 6.5 (phosphate ester hydrolytic reaction.

(4) pH stability: pH 3.5 to 12.0 (treatment at 30° C. for 60 minutes).

(5) Optimum temperature: about 35° C.

(6) Temperature stability: stable up to 40° C. (treatment at pH 7.0 for 30 minutes).

(7) Effect of the addition of metal ion and inhibitor: This enzyme exhibits no activation phenomenon relevant to its activity by addition of any metal ion The activity is inhibited by Fe 2+ , Ag 2+ , Pb 2+ , Hg 2+ , and Cu 2+ . The activity is also inhibited by iodoacetic acid.

(8) Molecular weight: The calculated molecular weight is about 188,000 in accordance with high-performance liquid chromatography (TSKgel G-3000SW, produced by Toyo Soda).

(9) Subunit molecular weight: The calculated subunit molecular weight is about 24,500 in accordance with SDS-polyacrylamide gel electrophoresis.

This enzyme also exhibits not only the activity to transfer phosphate group to a nucleoside, but also the activity to reversely hydrolyze a phosphate ester, in the same manner as the enzyme purified from the cell-free extract of Morganella morganii NCIMB 10466. Moreover, this enzyme exhibits the phosphate ester hydrolytic activity (phosphomonoesterase activity) which is higher than the transphosphorylation activity by not less than 30 times. Accordingly, it has been determined that this enzyme is an acid phosphatase.

Sodium pyrophosphate (15 g/dl) and inosine (3 g/dl) were dissolved in sodium acetate buffers each having pH of 5.5, 5.0, 4.5, 4.0, and 3.5, to which the enzyme sample described above was added so that a concentration of 50 units/dl was obtained. The reaction mixture was incubated at 30° C. for 6 hours while maintaining each pH, and the amount of produced 5′-inosinic acid was measured along with passage of time. Produced inosinic acid contained only 5′-inosinic acid. By-production of 2′-inosinic acid and 3′-inosinic acid was not observed at all. The result is shown in FIG. 2 . The velocity of 5′-inosinic acid production was maximum at pH 5.0. However, the maximum accumulated amount of 5′-inosinic acid was higher at lower pH. The reaction condition at pH 4.0 was most efficient for production of 5′-inosinic acid. 5′-Inosinic acid was produced and accumulated in an amount of 1.56 g/dl by performing the reaction at 30° C. at pH 4.0 for 3 hours.

Phosphorylation Reaction of Various Nucleosides by Acid Phosphatase Sample Derived from Escherichia blattae

Sodium pyrophosphate (15 g/dl) and inosine, guanosine, uridine, or cytidine (3 g/dl) were dissolved in sodium acetate buffer (pH 4.0), to which the enzyme sample prepared in Example 4 was added so that its concentration was 50 units/dl. The reaction mixture was incubated at 35° C. for 3 hours while maintaining pH at 4.0. The amount of produced nucleoside-5′-ester is shown in Table 5.

Produced nucleotide contained only nucleoside-5′-ester. By-production of nucleoside-2′-ester and nucleoside-3′-ester was not observed at all.

Production of 5′-Inosinic acid from Various Phosphate Compounds as Phosphate Group Donors by Acid Phosphatase Sample Derived from Escherichia blattae

Inosine (2 g/dl) and sodium tripolyphosphate, sodium polyphosphate (trade name: Polygon P, produced by Chiyoda Chemical), disodium phenylphosphate, or disodium carbamyl phosphate (10 g/dl) as a phosphate group donor were dissolved in sodium acetate buffer (pH 4.0), to which the enzyme sample prepared in Example 4 was added so that its concentration was 50 units/dl. The reaction mixture was incubated at 35° C. for 3 hours while maintaining pH at 4.0. The amount of produced 5′-inosinic acid is shown in Table 6.

5′-Inosinic acid was efficiently produced and accumulated by using any of the phosphate group donors. However, the accumulated amount of 5′-inosinic acid was the highest when sodium polyphosphate was used as the phosphate group donor.

Isolation of Gene Coding for Acid Phosphatase from Chromosome of Morganella morganii

(1) Determination of N-terminal Amino Acid Sequence

The acid phosphatase purified from the cell-free extract of Morganella morganii NCIMB 10466 in accordance with the method described in Example 1 was adsorbed to DITC membrane (produced by Milligen/Biosearch), and its N-terminal amino acid sequence was determined by using Prosequencer 6625 (produced by Milligen/Biosearch). An N-terminal amino acid sequence comprising 20 residues shown in SEQ ID NO: 1 in the Sequence Listing was determined.

(2) Isolation of DNA Fragment Containing Gene Coding for the Acid Phosphatase

Chromosomal DNA was extracted from cultivated microbial cells of Morganella morganii NCIMB 10466 in accordance with a method of Murray and Thomson ( Nucl. Acid Res., 4321, 8 (1980)). The chromosomal DNA was partially degraded with restriction enzyme Sau3AI. After that, DNA fragments of 3 to 6 kbp were fractionated by means of sucrose density gradient centrifugation. A plasmid vector pUC118 (produced by Takara Shuzo) was digested with restriction enzyme BamHI, which was ligated with the partially degraded chromosomal DNA fragments. DNA ligation was performed by using DNA ligation kit (produced by Takara Shuzo) in accordance with a designated method. After that, Escherichia coli JM109 (produced by Takara Shuzo) was transformed with an obtained DNA mixture in accordance with an ordinary method. Transformants were plated on an L agar medium containing 100 μg/ml of ampicillin, and they were grown to prepare a gene library.

A reaction solution containing 4 mM p-nitrophenylphosphoric acid and 100 mM MES/NaOH buffer (pH 6.5) was poured onto a surface of the agar medium on which the transformants had grown, and the temperature was kept at 30° C. for 15 minutes. Strains which had expressed the phosphatase activity liberated p-nitrophenol and exhibited a yellow color. Accordingly, transformants were selected by using this phenomenon as an index. As a result of screening for a gene expression library comprising about 20,000 strains of transformants, 30 strains of transformants which had expressed the phosphatase activity were obtained.

The transformants (30 strains), which had expressed the phosphatase activity, were subjected to single colony isolation. Single colonies were inoculated to an L-medium (2.5 ml) containing 100 μg/ml of ampicillin, and they were cultivated at 37° C. for 16 hours. Sodium acetate buffer (100 mM, pH 5.0, 50 1) containing inosine (2 g/dl) and sodium pyrophosphate (10 g/dl) was added to microbial cells harvested from culture, and the reaction mixture was incubated at 30° C. for 16 hours. Production of 5′-inosinic acid was detected by HPLC analysis to select microbial strains having the transphosphorylation activity. As a result, we succeeded in obtaining 5 strains of transformants which exhibited the transphosphorylation activity and which were assumed to harbor a DNA fragment containing the objective acid phosphatase gene were obtained.

Determination of Nucleotide Sequence of Acid Phosphatase Gene Derived from Morganella morganii NCIMB 10466

The inserted DNA fragment was analyzed by preparing a plasmid in accordance with an alkaline lysis method ( Molecular Cloning 2nd edition (J. Sambrook, E. F. Fritsch and T. Maniatis, Cold Spring Harbour Laboratory Press, pl. 25 (1989)) from one strain of the transformants which were assumed to harbor the DNA fragment containing the acid phosphatase gene derived from Morganella morganii NCIMB 10466 obtained in Example 7. This plasmid was designated as pMPI501. FIG. 3 shows a determined restriction enzyme map of the inserted DNA fragment.

The region of the acid phosphatase gene was further specified by subcloning. As a result, it was suggested that this acid phosphatase gene was contained in a fragment having a size of 1.2 Kbp excised by restriction enzymes HindIII and EcoRI. Thus in order to determine the nucleotide sequence, plasmid DNA was constructed in which the fragment of 1.2 kbp was ligated with pUC118 having been digested with HindIII and EcoRI. Escherichia coli JM109 (produced by Takara Shuzo) was transformed with this plasmid DNA designated as pMPI505 in accordance with an ordinary method, which was plated on an L agar medium containing 100 μg/ml of ampicillin to obtain a transformant.

The plasmid was extracted in accordance with the alkaline lysis method from the transformant of Escherichia coli JM109 (produced by Takara Shuzo) harboring pMPI505 to determine the nucleotide sequence. The nucleotide sequence was determined in accordance with the method of Sanger ( J. Mol. Biol., 143, 161 (1980)) by using Taq DyeDeoxy Terminator Cycle Sequencing Kit (produced by Applied Biochemical). A nucleotide sequence of a determined open reading frame is shown in SEQ ID NO: 2 in the Sequence Listing. An amino acid sequence of the protein deduced from the nucleotide sequence is shown in SEQ ID NO: 3 in the Sequence Listing. A partial sequence, which was completely coincident with the N-terminal amino acid sequence of the purified enzyme, was found in the amino acid sequence. The N-terminal of the purified enzyme starts from the 21th alanine residue of the sequence shown in SEQ ID NO: 3. Accordingly, it is assumed that the amino acid sequence shown in SEQ ID NO: 3 is that of a precursor protein, and that a peptide comprising a range from the 1st methionine residue to the 20th alanine residue is eliminated after translation. An amino acid sequence of a mature protein thus deduced is shown in SEQ ID NO: 4 in the Sequence Listing. A molecular weight of the mature protein estimated from the amino acid sequence is calculated to be 24.9 kilodaltons, which is well coincident with the result of SDS-PAGE for the purified enzyme. According to the results described above, and because of the fact that the transformant harboring the plasmid containing this fragment exhibited the transphosphorylation activity, it was identified that this open reading frame was the region coding for the objective acid phosphatase.

The nucleotide sequence and the amino acid sequence were respectively compared with known sequences for homology. Data bases of EMBL and SWISS-PROT were used. As a result, it has been revealed that the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing is coincident with a nucleotide sequence of a known acid phosphatase gene derived from Morganella morganii (Thaller, M. C. et al., Microbiology, 140, 1341 (1994)) except that 54th G is A, 72th G is A, 276th T is G, 378th T is C, 420th G is T, 525th C is G, 529th C is T, and 531th G is A in the latter, and that the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing is the same as that of the acid phosphatase gene derived from Morganella morganii . Namely, the gene, which codes for the protein comprising the amino acid sequence shown in SEQ ID NO: 4 in the Sequence Listing, is the acid phosphatase gene of Morganella morganii NCIMB 10466.

A precursor protein comprises 249 amino acids, and a molecular weight of the protein deduced from its sequence is 27.0 kilodaltons.

The strain of Escherichia coli JM109 transformed by a plasmid pMPI505, has been designated as AJ13143, which has been internationally deposited on Feb. 23, 1996 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan) under the provisions of the Budapest Treaty, and awarded a deposition number of FERM BP-5422.

Amplification of Activity by Expressing Gene of Acid Phosphatase Derived from Morganella morganii NCIMB 10466

Escherichia coli JM109/pMPI505 constructed in Example 8 was inoculated to an L-medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and it was cultivated at 37° C. for 16 hours. Microbial cells were harvested from its culture by centrifugation, and they were washed once with physiological saline. The microbial cells were suspended in 100 mM potassium phosphate buffer (5 ml, pH 7.2), and they were disrupted by means of a ultrasonic treatment performed at 4° C. for 20 minutes. The treated solution was centrifuged to remove an insoluble fraction, and thus a cell-free extract was prepared.

The transphosphorylation activity of the obtained cell-free extract was measured while using controls of cell-free extracts prepared from the wild type strain of Morganella morganii and Escherichia coli JM109 transformed with the plasmid pUC118 in the same manner as described above. The result is shown in Table 7. The transphosphorylation activity was not detected in Escherichia coli JM109/pUC118. The transphosphorylation activity was also low in the wild type strain of Morganella morganii . On the other hand, Escherichia coli JM109/pMPI505 exhibited a high transphosphorylation activity which was 150 times as high as that of the wild type stain of Morganella morganii in specific activity. According to the result, it has been demonstrated that the introduced DNA fragment allows Escherichia coli to express the acid phosphatase at a high level.

FIG. 4 illustrates the amount of 5′-ionsinic acid produced in a reaction performed with a strain harboring an acid phosphatase gene derived from Morganella morganii.

Isolation of Gene Coding for Acid Phosphatase from Chromosome of Escherichia blattae

(1) Determination of the N-terminal Amino Acid Sequence

The acid phosphatase purified from the cell-free extract of Escherichia blattae JCM 1650 was adsorbed to DITC membrane (produced by Milligen/Biosearch), and its N-terminal amino acid sequence was determined by using Prosequencer 6625 (produced by Milligen/Biosearch). An N-terminal amino acid sequence comprising 15 residues shown in SEQ ID NO: 8 in the Sequence Listing was determined.

(2) Isolation of DNA Fragment Containing Gene Coding for Acid Phosphatase

Chromosomal DNA was extracted from cultivated cells of Escherichia blattae JCM 1650 in accordance with a method of Murray and Thomson ( Nucl. Acid Res., 4321, 8 (1980)). The chromosomal DNA was partially degraded with Sau3AI. After that, DNA fragments of 3 to 6 kbp were fractionated by means of sucrose density gradient centrifugation. A plasmid vector pUC118 (produced by Takara Shuzo) was digested with BamHI, which was ligated with the partially degraded chromosomal DNA fragments. DNA ligation was performed by using DNA ligation kit (produced by Takara Shuzo) in accordance with a designated method. After that, Escherichia coli JM109 (produced by Takara Shuzo) was transformed with an obtained DNA mixture in accordance with an ordinary method. Transformants were plated on an L agar medium containing 100 μg/ml of ampicillin, and they were grown to prepare a gene library.

A reaction solution containing 4 mM p-nitrophenylphosphoric acid and 100 mM MES/NaOH buffer (pH 6.5) was poured onto a surface of the agar medium on which the transformants had grown, and the temperature was kept at 30° C. for 15 minutes. Strains which had expressed the phosphatase activity liberated p-nitrophenol and exhibited a yellow color. Accordingly, transformants were selected by using this phenomenon as an index. As a result of screening for a chromosomal gene expression library comprising about 8,000 strains of transformants, 14 strains of transformants which had expressed the phosphatase activity were obtained.

The transformants (14 strains), which had expressed the phosphatase activity, were subjected to single colony isolation. Single colonies were inoculated to an L-medium (2.5 ml) containing 100 μg/ml of ampicillin, and they were cultivated at 37° C. for 16 hours. Sodium acetate buffer (100 mM, pH 5.0, 50 1) containing inosine (2 g/dl) and sodium pyrophosphate (10 g/dl) was added to microbial cells harvested from culture liquids to perform the reaction at 30° C. for 16 hours. Production of 5′-inosinic acid was detected by HPLC analysis to select strains having the transphosphorylation activity. As a result, 3 strains of transformants which exhibited the transphosphorylation activity and which were assumed to harbor a DNA fragment containing the objective acid phosphatase gene were obtained.

Determination of Nucleotide Sequence of Acid Phosphatase Gene Derived from Escherichia blattae JCM 1650

The inserted DNA fragment was analyzed by extracting a plasmid in accordance with the alkaline lysis method from one strain of the transformants which were assumed to harbor the DNA fragment containing the acid phosphatase gene derived from Escherichia blattae JCM 1650 obtained in Example 11. This plasmid was designated as pEPI301. FIG. 5 shows the determined restriction enzyme map of the inserted DNA fragment.

The region of the acid phosphatase gene was further specified by subcloning. As a result, it was suggested that this acid phosphatase gene was included in a fragment having a size of 2.4 Kbp excised by restriction enzymes ClaI and BamHI. Thus in order to determine the nucleotide sequence, plasmid DNA was constructed in which the fragment was ligated with pBluescript KS(+) (produced by Stratagene) having been digested with ClaI and BamHI. Escherichia coil JM109 (produced by Takara Shuzo) was transformed with the plasmid DNA designated as pEPI305 in accordance with an ordinary method, which was plated on an L agar medium containing 100 μg/ml of ampicillin to obtain a transformant.

The plasmid was extracted in accordance with the alkaline lysis method from the transformant of Escherichia coli JM109 (produced by Takara Shuzo) harboring pEPI305 to determine the nucleotide sequence. A nucleotide sequence of a determined open reading frame is shown in SEQ ID NO: 6 in the Sequence Listing. An amino acid sequence of the protein deduced from the nucleotide sequence is shown in SEQ ID NO: 7 in the Sequence Listing. A partial sequence, which was completely coincident with the N-terminal amino acid sequence of the purified enzyme, was found in the amino acid sequence. The N-terminal of the purified enzyme starts from the 19th leucine residue of the sequence shown in SEQ ID NO: 7. Accordingly, it is assumed that the amino acid sequence shown in SEQ ID NO: 7 is that of a precursor protein and that a peptide comprising a range from the 1st methionine residue to the 18th alanine residue is eliminated after translation. An amino acid sequence of a mature protein thus deduced is shown in SEQ ID NO: 8 in the Sequence Listing. Accordingly, an estimated molecular weight of the mature protein is calculated to be 25.1 kilodaltons, which is well coincident with the result of SDS-PAGE for the purified enzyme. According to the results described above, and because of the fact that the transformant harboring the plasmid containing this fragment exhibited the transphosphorylation activity, it was identified that this open reading frame was the region coding for the desired acid phosphatase.

Namely, the gene, which codes for the protein comprising the amino acid sequence shown in SEQ ID NO: 8 in the Sequence Listing, is the acid phosphatase gene of Escherichia blattae JCM 1650.

The nucleotide sequence and the amino acid sequence were respectively compared with known sequences for homology. Data bases of EMBL and SWISS-PROT were used. As a result, it has been revealed that the protein shown in SEQ ID NO: 8 and DNA coding for it are novel. A precursor protein encoded by this gene comprises 249 amino acids, and a molecular weight of the protein deduced from its sequence is 27.0 kilodaltons.

The amino acid sequence was compared with known sequences respectively for homology. As a result, this protein exhibited a high degree of homology with the acid phosphatase of Providencia stuartii (77.1%) with the acid phosphatase of Morganella morganii in Example 8 (77.1%), and with acid phosphatase of Salmonella typhimurium (44.3%).

The strain of Escherichia coli JM109 transformed by a plasmid pEPI305, has been designated as AJ13144, which has been internationally deposited on Feb. 23, 1996 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) under the provisions of the Budapest Treaty, and awarded a deposition number of FERM BP-5423.

Amplification of Activity by Expressing Gene of Acid Phosphatase Derived from Escherichia blattae JCM 1650

Escherichia coli JM109/pEPI305 constructed in Example 12 was inoculated to an L medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and it was cultivated at 37° C. for 16 hours. Microbial cells were harvested from its culture by centrifugation, and they were washed once with physiological saline. The microbial cells were suspended in 100 mM potassium phosphate buffer (5 ml, pH 7.2), and were disrupted by means of a ultrasonic treatment performed at 4° C. for 20 minutes. The treated solution was centrifuged to remove an insoluble fraction, and thus a cell-free extract was prepared.

The transphosphorylation activity of the obtained cell-free extract was measured while using controls of cell-free extracts prepared from the wild type strain of Escherichia blattae and Escherichia coli JM109 transformed with the plasmid pBluescript KS(+) in the same manner as described above. The result is shown in Table 8. The transphosphorylation activity was not detected in Escherichia coli JM109/pBluescript KS(+). The transphosphorylation activity was also low in the wild type strain of Escherichia blattae . On the other hand, Escherichia coli JM109/pEPI305 exhibited a high transphosphorylation activity which was 120 times as high as that of the wild type strain of Escherichia blattae in specific activity. According to the result, it has been demonstrated that the introduced DNA fragment allows Escherichia coli to express the acid phosphatase at a high level.

Production of 5′-Inosinic Acid from Inosine by Using Strain Harboring Acid Phosphatase Gene Derived from Escherichia blattae JCM 1650

Sodium pyrophosphate (12 g/dl) and inosine (6 g/dl) were dissolved in 100 mM sodium acetate buffer (pH 4.0), to which the microbial cells of Escherichia coli JM109/pEPI305 described above were added to give a cell concentration of 200 mg/dl as converted into a dry weight of the microbial cells. The reaction mixture was incubated at 35° C. for 10 hours while maintaining pH at 4.0, and the amount of produced 5′-inosinic acid was measured along with passage of time. Produced inosinic acid contained only 5′-inosinic acid. By-production of 2′-inosinic acid and 3′-inosinic acid was not observed at all. The result is shown in FIG. 6. 5′-Inosinic acid was produced and accumulated extremely efficiently in a short period of time in the reaction to produce 5′-inosinic acid from pyrophosphate and inosine by using this microorganism.

Preparation of a Gene Encoding An Acid Phosphatase with Lowered Phosphomonoesterase Activity

As described in Examples 13 and 14, the strain harboring the acid phosphatase gene derived from Escherichia blattae expresses a considerable amount of the acid phosphatase, and 5′-inosinic acid is produced and accumulated extremely efficiently in a short period of time in the reaction to produce 5′-inosinic acid from pyrophosphate and inosine by using this microorganism. However, it has been revealed that the accumulated amount of 5′-inosinic acid does not exceed a certain degree because produced 5′-inosinic acid undergoes degradation by the phosphomonoesterase activity possessed by the acid phosphatase itself. Thus the enzyme was intended to be improved by introducing mutation into the acid phosphatase gene derived from Escherichia blattae cloned in Example 11, in accordance with the site-directed mutagenesis method by using PCR.

Oligonucleotides MUT300, MUT310, and MUT320 shown in SEQ ID NO: 9, 10, and 11 in the Sequence Listing were synthesized respectively in accordance with the phosphoamidite method by using a DNA synthesizer (Model 394 produced by Applied Biosystems).

The plasmid pEPI305 (1 ng) as a template prepared in Example 12, M13 primer RV (produced by Takara Shuzo) and MUT310 oligonucleotide (each 2.5 mol) as primers, and Taq DNA polymerase (2.5 units, produced by Takara Shuzo) were added to 100 mM Tris-HCl buffer (pH 8.3, 100 μl) containing dATP, dCTP, dGTP, dTTP (each 200 μM), potassium chloride (50 mM), and magnesium chloride (1.5 mM) to perform a PCR reaction in which a cycle comprising periods of 30 seconds at 94° C., 2 minutes at 55° C., and 3 minutes at 72° C. was repeated 25 times. The PCR reaction was performed by using Thermal Cycler PJ2000 type (produced by Takara Shuzo). Also, a PCR reaction was performed in the same manner as described above by using plasmid pEPI305 (1 ng) as a temperate, and M13 primer M3 (produced by Takara Shuzo) and MUT300 oligonucleotide (each 2.5 μmol) as primers. Each of the reaction solutions was purified by gel filtration to remove the primers by using Microspin column S-400 (produced by Pharmacia).

Each of the PCR reaction products (1 μl) was added to 100 mM Tris-HCl buffer (pH 8.3, 95 μl) containing dATP, dCTP, dGTP, dTTP (each 200 μM), potassium chloride (50 mM), and magnesium chloride (1.5 mM), and it was heated at 94° C. for 10 minutes, followed by cooling to 37° C. over 60 minutes. After that, the temperature was kept at 37° C. for 15 minutes to form a heteroduplex. Taq DNA polymerase (2.5 units) was added thereto to perform a reaction at 72° C. for 3 minutes so that the heteroduplex was completed. After that, M13 primer RV and M13 primer M3 (each 2.5 μmol) were added to this reaction solution to perform a PCR reaction in which a cycle comprising periods of 30 seconds at 94° C., 2 minutes at 55° C., and 3 minutes at 72° C. was repeated 10 times.

A product of the second PCR reaction was digested with ClaI and BamHI followed by phenol/chloroform extraction and ethanol precipitation. This DNA fragment was ligated with pBluescript KS(+) having been digested with ClaI and BamHI. Escherichia coli JM109 (produced by Takara Shuzo) was transformed with obtained plasmid DNA in accordance with an ordinary method, which was plated on an L agar medium containing 100 μg/ml of ampicillin to obtain a transformant.

The plasmid was extracted from the transformant in accordance with the alkaline lysis method to determine its nucleotide sequence, confirming that the desired nucleotide was substituted. Thus a mutant gene coding for a mutant phosphatase was prepared in which the 74th glycine residue (GGG) of the mature protein was substituted with an aspartic acid residue (G*A*T). The plasmid containing this mutant gene was designated as pEPI310.

A mutant gene coding for a mutant phosphatase was prepared in which the 153th isoleucine residue (ATC) of the mature protein was substituted with a threonine residue (A*CC), in accordance with the same procedure as described above by using pEPI305 as a template, and MUT300 and MUT320 oligonucleotides as primers. The plasmid containing this mutant gene was designated as pEPI320. Further, a mutant gene coding for a mutant phosphatase was prepared in which the 74th glycine residue (GGG) of the mature protein was substituted with an aspartic acid residue (G*A*T), and the 153th isoleucine residue (ATC) of the mature protein was substituted with a threonine residue (A*CC), in accordance with the same procedure as described above by using pEPI310 as a template, and MUT300 and MUT320 oligonucleotides as primers. The plasmid containing this mutant gene was designated as pEPI330.

Escherichia coli JM109/pEPI310 , Escherichia coli JM109/pEPI320, and Escherichia coli JM109/pEPI330 into which the plasmids containing the respective mutant acid phosphatase genes had been introduced, and Escherichia coli JM109/pEPI305 into which the plasmid containing the wild type acid phosphatase gene had been introduced were inoculated to an L medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and they were cultivated at 37 C. for 16 hours. Microbial cells were harvested from their culture, and they were washed once with physiological saline. The microbial cells were suspended in 100 mM potassium phosphate buffer (5 ml, pH 7.0), and they were disrupted by means of a ultrasonic treatment performed at 4 C. for 20 minutes. The treated solutions were centrifuged to remove insoluble fractions, and thus cell-free extracts were prepared. Phosphomonoesterase activities and transphosphorylation activities of the obtained cell-free extracts were measured at pH 4.0, and they were compared with an activity of the wild strain.

Table 9 shows the result of measurement of phosphomonoesterase activities and transphosphorylation activities of wild type acid phosphatase and acid phosphatases with lowered phosphomonoesterase activity. It shows that both of phosphomonoesterase activities and transphosphorylation activities of acid phosphatases with lowered phosphomonoesterase activity are lowered as compared with wild type acid phosphatase, and that degrees of decrease of phosphomonoesterase activities are larger than that of transphosphorylation activities, with the result that the ratio of phosphomonoesterase activity to transphosphorylation activity of the mutant acid phosphatase is lowered in comparison with the wild type acid phosphatase.

Production of 5′-Inosinic Acid from Inosine by Using the Strains Harboring a Gene Encoding the Acid Phosphatase with Lowered Phosphomonoesterase Activity

Escherichia coli JM109/pEPI310 , Escherichia coli JM109/pEPI320, and Escherichia coli JM109/pEPI330 into which the plasmids containing the genes encoding the acid phosphatases with lowered phosphomonoesterase activity had been introduced, and Escherichia coil JM109/pEPI305 into which the plasmid containing the wild type acid phosphatase gene had been introduced were inoculated to an L medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and they were cultivated at 37° C. for 16 hours.

Sodium pyrophosphate (12 g/dl) and inosine (6 g/dl) were dissolved in sodium acetate buffer (pH 4.0), to which microbial cells of each of the strains of Escherichia coil obtained by the cultivation described above were added to give a cell concentration of 200 mg/dl as converted into a dry weight of the microbial cells. The reaction mixture was incubated at 35° C. for 32 hours while maintaining pH at 4.0, and the amount of produced 5′-inosinic acid was measured along with passage of time. The result is shown in FIG. 7 .

In FIG. 7, the axis of ordinate indicates the concentration of 5′-inosinic acid (mg/dl), and the axis of abscissa indicates the reaction time (h). Progress of the reaction is indicated by solid circles for Escherichia coli JM109/pEPI305, solid triangles for Escherichia coli JM109/pEPI310, blanked circles for Escherichia coli JM109/pEPI320, and blanked squares for Escherichia coil JM109/pEPI330, as measured by using the cells of the respective strains.

The velocity of degradation of produced 5′-inosinic acid was decreased in the reaction to produce 5′-inosinic acid from inosine by using the stains harboring the acid phosphatase with lowered phosphomonoesterase activity. As a result, the yield and the accumulated amount of 5′-inosinic acid were increased. The highest accumulation of 5′-inosinic acid was exhibited by Escherichia coli JM109/pEPI330 as the strain harboring the gene encoding the acid phosphatase with lowered phosphomonoesterase activity in which the 74th glycine residue and the 153th isoleucine residue were substituted with the aspartic acid residue and the threonine residue respectively.

Production of Various Nucleoside-5′-Phosphate Esters by Using the Strains Horboring a Gene Encoding the Acid Phosphatase with Lowered Phosphomonoesterase Activity

Escherichia coli JM109/pEPI330 into which the plasmid containing the gene encoding the acid phosphatase with lowered phosphomonoesterase activity had been introduced was inoculated to an L medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and it was cultivated at 37° C. for 16 hours.

Sodium pyrophosphate (12 g/dl), and inosine, guanosine, uridine, or cytidine (6 g/dl) as a phosphate group acceptor were dissolved in 100 mM sodium acetate buffer (pH 4.0), to which the microbial cells described above were added to give a cell concentration of 200 mg/dl as converted into a dry weight of the cells. The reaction mixture was incubated at 35° C. for 32 hours while maintaining pH at 4.0. Amounts of produced nucleoside-5′-phosphate esters are shown in Table 10. Produced nucleotide contained only nucleoside-5′-phosphate ester. By-production of nucleoside-2′-phosphate ester and nucleoside-3′-phosphate ester was not observed at all.

Production of 5′-Inosinic Acid from Various Phosphate Compounds as Phosphate Group Donors by Using the Strains Horboring a Gene Encoding the Acid Phosphatase with Lowered Phosphomonoesterase Activity

Escherichia coli JM109/pEPI330 into which the plasmid containing the mutant acid phosphatase gene had been introduced was inoculated to an L medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and it was cultivated at 37° C. for 16 hours.

Inosine (6 g/dl) and sodium tripolyphosphate, sodium polyphosphate (trade name: Polygon P, produced by Chiyoda Chemical), disodium phenylphosphate, or disodium carbamyl phosphate (12 g/dl) as a phosphate group donor were dissolved in 100 mM sodium acetate buffer (pH 4.0), to which the microbial cells described above were added to give a cell concentration of 200 mg/dl as converted into a dry weight of the cells. The reaction mixture was incubated at 35° C. for 32 hours while maintaining pH at 4.0. The amount of produced 5′-inosinic acid is shown in Table 11. 5′-Inosinic acid was efficiently produced and accumulated by using any of the phosphate group donors. However, the accumulated amount of 5′-inosinic acid was the highest when polyphosphoric acid was used as the phosphate group donor.

Studies on Production of a New Mutant Acid Phosphatase Gene Derived from E. blattae JCM1650 and Enzymological Properties of the Mutant Acid Phosphatase Gene

In Examples 19 to 22, the transphosphorylation activity to a nucleoside was measured under the following conditions. The reaction was conducted at 30° C. and a pH of 4.0 for 10 minutes using 1 ml of a reaction solution containing 40 μml/ml of inosine, 100 μmol/ml of sodium pyrophosphate, 100 μmol/ml of a sodium acetate buffer (pH 4.0) and an enzyme. This reaction was terminated with the addition of 200 μl of 2-N hydrochloric acid. Then, the precipitate was removed through centrifugation, and the amount of 5′-inosinic acid formed through the transphosphorylation was determined under the above-mentioned conditions. The amount of the enzyme with which to produce 1 μmol of inosinic acid under these standard reaction conditions was defined as 1 unit.

Further, the transphosphorylation activity was measured by changing the inosine concentration from 10 to 100 μmol/ml under the reaction conditions of the above-mentioned composition, and the rate constant of inosine in the transphosphorylation activity was determined using the Hanes-Woolf plot [ Biochem. J., 26, 1406 (1932), incorporated herein by reference].

As described below, the detailed analysis was conducted with respect to the mutant enzyme by which to improve the productivity of the nucleoside-5′-phosphate ester described in Example 15. Consequently, it was found that the affinity for nucleoside of the mutant enzyme was improved by 2 times as compared with that of the wild-type enzyme. Therefore, the present inventors considered that the productivity of the nucleoside-5′-phosphate ester could be improved by increasing the affinity for nucleoside of the above-mentioned enzyme, and they further modified the enzyme by the genetic engineering method.

Plasmid pEPI305 containing the gene encoding the wild-type acid phosphatase derived from E. blattae described in Example 15 was used, and the site specific mutation was introduced into this plasmid DNA by the genetic engineering method to produce a gene encoding the mutant acid phosphatase. pEPI305 is a plasmid DNA formed by binding a DNA fragment of 2.4 Kbp cleaved with restriction endonucleases ClaI and BamHI and containing a gene encoding a wild-type acid phosphatase derived from E. blattae JCM1650 to pBluescript KS(+) (supplied by Stratagene) cleaved with ClaI and BamHI. The base sequence of the gene encoding the acid phosphatase is represented by SEQ ID NO: 6 in the Sequence Listing. Further, an amino acid sequence of a precursor protein anticipated from this base sequence is represented by SEQ ID NO: 7 in the Sequence Listing. From the analytical results of the purified enzyme (described in Example 4), the amino acid sequence of the maturation protein is presumed to be represented by SEQ ID NO: 8 in the Sequence Listing.

Oligonucleotides MUT300 (SEQ ID NO: 9 in the Sequence Listing), MUT310 (SEQ ID NO: 10 in the Sequence Listing), MUT320 (SEQ ID NO: 11 in the Sequence Listing), MUT330 (SEQ ID NO: 12 in the Sequence Listing), MUT340 (SEQ ID NO: 13 in the Sequence Listing), MUT350 (SEQ ID NO: 14 in the Sequence Listing), MUT360 (SEQ ID NO: 15 in the Sequence Listing), MUT370 (SEQ ID NO: 16 in the Sequence Listing), MUT380 (SEQ ID NO: 17 in the Sequence Listing) and MUT390 (SEQ ID NO: 18 in the Sequence Listing) were synthesized by the phosphoamidite method using a DNA synthesizer (Model 394 supplied by Applied Biosystem).

One nanogram of pEPI305 as a template, 2.5 mols of M13 Primer RV (supplied by Takara Shuzo Co., Ltd.), 2.5 μmols of oligonucleotide MUT310 and 2.5 units of tac DNA polymerase (supplied by Takara Shuzo Co., Ltd.) were added to 100 μl of a Tris-hydrochloride buffer (pH 8.3) containing 200 μM of dATP, 200 M of dCTP, 200 μM of dGTP, 200 μM of dTTP, 50 mM of potassium chloride and 1.5 mM of magnesium chloride. PCR was conducted in which a three-part step, namely, at 94° C. for 30 seconds, at 55° C. for 2 minutes and at 72° C. for 3 minutes was repeated 25 times. In this reaction, a thermal cycler PJ2000 model (supplied by Takara Shuzo Co., Ltd.) was used. Separately, PCR was likewise conducted using 1 ng of plasmid DNA pEPI305 as a template, 2.5 μmols of M13 Primer M3 (supplied by Takara Shuzo Co., Ltd.) as a primer and 2.5 μmols of oligonucleotide MUT300. Each of the reaction solutions was purified through gel filtration using a microspin column S-400 (supplied by Pharmacia) to remove the primer.

One microliter of each of the PCR solutions was added to 95 μl of a 100-mM Tris-hydrochloride buffer (pH 8.3) containing 200 μM of dATP, 200 μM of dCTP, 200 μM of dGTP, 200 μM of dTTP, 50 mM of potassium chloride and 1.5 mM of magnesium chloride. The mixture was heated at 94° C. for 10 minutes, then cooled to 37° C. over the course of 60 minutes, and warmed at 37° C. for 15 minutes to form a heteroduplex. To this were added 2.5 units of tac DNA polymerase, and the reaction was conducted at 72° C. for 3 minutes to complete the heteroduplex. Subsequently, 2.5 μmols of M13 Primer RV and 2.5 μmols of M13 Primer M3 were added to the reaction solution, and PCR was conducted in which a three-part step, namely, at 94° C. for 30 seconds, at 5° C. for 2 minutes and at 72° C. for 3 minutes was repeated 10 times.

The second PCR product was cleaved with ClaI and BamHI, then extracted with a mixture of phenol and chloroform, and precipitated with ethanol. This DNA fragment was bound to pBluescript KS (+) cleaved with ClaI and BamHI. E. coli JM109 (supplied by Takara Shuzo Co., Ltd.) was transformed in a usual manner using the resulting plasmid DNA. This was plated on an L agar medium containing 100 μg/ml of ampicillin to obtain a transformant. A plasmid was prepared from the transformant by an alkali bacteriolysis method, the base sequence was determined, and it was identified that the desired base was substituted. The determination of the base sequence was conducted by the method of Sanger et al. [ J. Mol. Biol., 143, 161 (1980)] using a Taq DyeDeoxy Terminator Cycle Sequencing Kit (supplied by Applied Biochemical). In this manner, the mutant gene encoding the mutant phosphatase in which the 74th glycine residue (GGG) of the maturation protein was substituted with the aspartic acid residue (G*A*T) was produced. This mutant gene-containing plasmid was designated pEPI310 (Example 15).

The above-mentioned procedure was repeated using the plasmid having the mutation introduced therein as a template to cumulatively introduce the site-specific mutation. A plasmid was produced from the transformant by the alkali bacteriolysis method, the base sequence was determined, and it was identified that the desired base was substituted. The resulting mutant genes encoding the mutant phosphatase and the mutation sites are shown in Table 12. The amino acid residue in the mutation site indicates an amino acid residue in the amino acid sequence of the mature protein represented by SEQ ID NO: 8 in the Sequence Listing.

Each of E. coli JM109/pEPI330 , E. coli JM109/pEPI340 , E. coli JM109/pEPI350 , E. coli JM109/pEPI360 , E. coli JM109/pEPI370 , E. coli JM109/pEPI380 , E. coli JM109/pEPI390 and E. coli JM109/pEPI400 each having introduced therein a plasmid containing the mutant acid phosphatase gene and E. coli JM109/pEPI305 having introduced therein a plasmid containing a wild-type acid phosphatase gene was inoculated into 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37° C. for 16 hours. The cells were collected from 2 liters of the culture solution of each of the strains through centrifugation, and washed once with a physiological saline solution. The cells were suspended in 50 ml of a 100-mM phosphate buffer (pH 7.0), and sonicated at 4° C. for 20 minutes to disrupt the cells. The thus-treated solution was centrifuged to remove insoluble fractions and prepare a cell-free extract. Each of the acid phosphatases was purified from each of the cell-free extracts by the method described in Example 4. Each of the enzyme products was uniform in the SDS-polyacrylamide electrophoresis.

The rate constant of inosine in the transphosphorylation of the purified mutant acid phosphatases and wild-type acid phosphatase was measured, and the results are shown in Table 13. It was found that the mutant enzyme expressed in E. coli JM109/pEPI330 having the improved productivity of the nucleoside-5′-phosphate ester as described in Example 15 has decreased V max but greatly decreased the K m value to inosine which means the increased affinity for inosine by twice or more as compared with the wild-type enzyme expressed in E. coli JM109/pEPI305. This suggested that the productivity of the nucleoside-5′-phosphate ester of this mutant enzyme was greatly improved not only because of the decrease in the nucleotidase activity but also because of the improvement in the affinity for nucleoside which was an important factor. Accordingly, it was expected that the increase in the affinity for nucleoside leads to the improvement in the productivity.

The new mutant enzymes expressed in E. coli JM109 having been introduced therein with the new mutant enzyme gene produced in this Example exhibited the affinity for inosine which was more improved than that of E. coli JM109/pEPI330 described in Example 15. Thus, it was expected that the productivity of the nucleoside-5′-phosphate ester was improved. Further, the mutant enzyme expressed in E. coli JM109/pEPI380 not only improved the affinity for inosine but also increased the V max value as compared with the wild-type enzyme. Still further, it was expected that the productivity of the nucleoside-5′-phosphate ester was improved.

Production of 5′-inosinic Acid using a New Mutant Acid Phosphatase Gene-Containing Strain

Each of E. coli JM109/pEPI330 , E. coli JM109/pEPI340 , E. coli JM109/pEPI360 , E. coli JM109/pEPI370 and E. coli JM109/pEPI380 each having introduced therein the plasmid containing the mutant acid phosphatase gene was inoculated into 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37° C. for 16 hours.

Pyrophosphoric acid (15 g/dl) and 8 g/dl of inosine were dissolved in an acetate buffer (pH 4.0). To the solution was added E. coli JM109 strain having introduced therein the above-mentioned mutant and wild-type acid phosphatase genes such that the concentration reached 200 mg/dl in terms of the dry cell weight. The reaction was conducted at 35° C. for 32 hours while maintaining the pH at 4.0, and the amount of 5′-inosinic acid formed over the course of time was measured. Inosinic acid formed was only 5′-inosinic acid, and the formation of 2′-inosinic acid and 3′-inosinic acid as by-products was not observed at all. The results are shown in FIG. 8 .

E. coli JM109/pEPI330 described in Example 15 showed the accumulation of 5′-inosinic acid in a large amount. Although the substrate still remained, the formation of 5′-inosinic acid stopped when the amount of 5′-inosinic acid accumulated reached 7.5 g/dl, and the amount of 5′-inosinic acid was no longer increased. By contrast, the new mutant acid phosphatase gene-containing strains provided the large amount of 5′-inosinic acid accumulated. Especially, in the reaction using E. coli JM109/pEPI370 and E. coli JM109/pEPI380, the larger amount of 5′-inosinic acid accumulated was provided. In addition, the reaction rate was high, showing that the productivity of 5′-inosinic acid was further improved greatly. In particular, in E. coli JM109/pEPI380, the reaction rate was high, and quite a high reactivity was shown.

Production of Various Nucleoside-5′-phosphate Esters using a New Mutant Acid Phosphatase Gene-Containing Strain

E. coli JM109/pEPI380 having introduced therein the plasmid containing the new mutant acid phosphatase gene was inoculated into 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37° C. for 16 hours.

Pyrophosphoric acid (15 g/dl) and 8 g/dl of inosine, guanine, uridine or cytidine as a phosphate acceptor were dissolved in a 100 mM acetate buffer (pH 4.5). To this was added the above-mentioned strain such that the concentration reached 100 mg/dl in terms of the dry cell weight. The reaction was conducted at 35° C. for 12 hours while maintaining the pH at 4.0. The amount of the nucleoside-5′-phosphate ester formed is shown in Table 14. The phosphorylation proceeded well with any of the nucleosides to form and accumulate the corresponding nucleoside-5′-phosphate esters. The nucleotide formed was only the nucleoside-5′-phosphate ester, and the formation of a nucleoside-2′-phosphate ester and a nucleoside-3′-phosphate ester as by-products was not observed at all.

Production of 5′-Inosinic Acid Using a New Acid Phosphatase Gene-Containing Strain and Various Phosphoric Acid Compounds as a Phosphate Donor

E. coli JM109/pEPI380 having introduced therein the plasmid containing the new mutant acid phosphatase gene was inoculated into 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37 C. for 16 hours.

Inosine (6 g/dl) and 15 g/dl of a tripolyphosphate, a polyphosphate (“Polygon P”, a trade name for a product of Chiyoda Kagaku K.K.), disodium phenylacetate or disodium carbamylphosphate were dissolved in a 100-mM acetate buffer (pH 4.0). To this was added the above-mentioned strain such that the concentration reached 100 mg/dl in terms of the dry cell weight. The reaction was conducted at 35° C. for 12 hours while maintaining the pH at 4.0. The amount of 5′-inosinic acid formed was shown in Table 15. 5′-Inosinic acid was formed and accumulated at good efficiency with any of the phosphate donors. Especially when a polyphosphate was used as a phosphate donor, 5′-inosinic acid was accumulated in the largest amount.

Isolation of Acid Phosphatase Gene Derived from Chromosome of Providencia stuartii and Determination of Nucleotide Sequence of the Gene

Oligonucleotides, PRP1 and PRP2, having nucleotide sequences illustrated in SEQ ID NO: 19 and 20 in the Sequence Listing, respectively, were synthesized. These oligonucleotides are designed to amplify a gene coding for acid phosphatase of Providencia stuartii on the basis of known nucleotide sequence of the gene coding for acid phosphatase of Providencia stuartii (Database of EMBL Accession number X64820).

Chromosomal DNA was extracted from cultivated microbial cells of Providencia stuartii ATCC 29851 in accordance with a method of Murray and Thomson ( Nucl. Acid Res., 4321, 8 (1980)). The chromosomal DNA (0.1 ng) as a template, oligonucleotides PRP1 and PRP2 (each 2.5 μmol) as primers, and Taq DNA polymerase (2.5 units, produced by Takara Shuzo) were added to 100 mM Tris-HCl buffer (pH 8.3, 100 1) containing dATP, dCTP, dGTP, dTTP (each 200 μM), potassium chloride (50 mM), and magnesium chloride (1.5 mM) to perform a PCR reaction in which a cycle comprising periods of 30 seconds at 94° C., 2 minutes at 55° C., and 3 minutes at 72° C. was repeated 30 times. The reaction solution was subjected to agarose gel electrophoresis, followed by recovering the amplified DNA fragment of about 1 kbp by means of glass powders (made by Takara Shuzo). The gene fragment was digested with BamHI, which was ligated with pUC118 digested with BamHI. The plasmid obtained as described above was designated as pPRP100.

Phosphomonoesterase activity and transphosphorylation activity of Escherichia coli JM109/pPRP100, a transformant to which pPRP100 was introduced, were measured. As a result, the strain showed an activity to transphosphorylate to nucleoside as well as phosphomonoesterase activity.

The plasmid was extracted in accordance with the alkaline lysis method from the transformant of Escherichia coli JM109/pPRP100 to determine the nucleotide sequence. A nucleotide sequence of a determined open reading frame and an amino acid sequence of the protein deduced from the nucleotide sequence are shown in SEQ ID NO: 21 and 22 in the Sequence Listing. The nucleotide sequence of the open reading frame is completely coincident with the nucleotide sequence of the known acid phosphatase gene of Providencia stuartii.

Isolation of Acid Phosphatase Genes Derived from Chromosomes of Enterobacter aerogenes, Klebsiella planticola and Serratia ficaria and Determination of Nucleotide Sequences of the Genes

Chromosomal DNA was extracted from cultivated microbial cells of Enterobacter aerogenes IFO 12010 , Klebsiella planticola IFO 14939 and Serratia ficaria IAM 13540 in accordance with a method of Murray and Thomson ( Nucl. Acid Res., 4321, 8 (1980)). Then, in accordance with the method described in Example 7(2), a chromosomal gene expression library comprising about 20,000 transformants of Escherichia coli JM109 was constructed and screened to obtain transformants which showed transphosphorylation activity. It was considered that each of these transformants harbors the acid phosphatase gene derived from each of the original strains.

Plasmid DNA was extracted from one of the transformants of Escherichia coli which was considered to have the acid phosphatase gene derived from Enterobacter aerogenes IFO 12010 in accordance with an alkaline lysis method and the inserted DNA of the plasmid was analyzed. The above plasmid was designated as pENP100. A restriction enzyme map of the inserted DNA derived from Enterobacter aerogenes IFO 12010 is shown in FIG. 9 .

As a result of specifying the region of acid phosphatase gene by subcloning, it was suggested that the acid phosphatase gene is contained in the 1.6 kbp fragment excised by restriction enzymes SalI and KpnI. Then, the SalI- KpnI fragment was ligated with pUC118 which was digested with SalI and KpnI to construct a plasmid. The resulting plasmid was designated as pENP110.

According to the procedure as described above, plasmid DNA was extracted from one of the transformants of Escherichia coil which was considered to have the acid phosphatase gene derived from Klebsiella planticola IFO 14939 in accordance with an alkaline lysis method and the insert DNA of the plasmid was analyzed. The above plasmid was designated as pKLP100. A restriction enzyme map of the inserted DNA derived from Klebsiella planticola IFO 14939 is shown in FIG. 10 .

As a result of specifying the region of acid phosphatase gene by subcloning, it was suggested that the acid phosphatase gene is contained in the 2.2 kbp fragment excised by restriction enzymes KpnI and EcoRI. Then, the KpnI- EcoRI fragment was ligated with pUC118 which was digested with KpnI and EcoRI to construct a plasmid. The resulting plasmid was designated as pKLP110.

Similarly, plasmid DNA was extracted from one of the transformants of Escherichia coil which was considered to have the acid phosphatase gene derived from Serratia ficaria IAM 13540 in accordance with an alkaline lysis method and the inserted DNA of the plasmid was analyzed. The above plasmid was designated as pSEP100. A restriction enzyme map of the inserted DNA derived from Serratia ficaria IAM 13540 is shown in FIG. 11 .

As a result of specifying the region of acid phosphatase gene by subcloning, it was suggested that the acid phosphatase gene is contained in the 1.4 kbp fragment excised by restriction enzymes HindIII. Then, the HindIII fragment was ligated with pUC118 which was digested with HindIII to construct a plasmid. The resulting plasmid was designated as pSEP110.

Then, the plasmid DNAs were extracted from the transformants, Escherichia coli JM109/pENP110 , Escherichia coli JM109/pKLP110 and Escherichia coli JM109/pSEP110, to which pENP110 pKLP110 and pSEP110 had been introduced, respectively, in accordance with an alkaline lysis method. The nucleotide sequences of inserts of these plasmids were determined in accordance with the method described in Example 8. The determined nucleotide sequences of open reading frames of the inserts are shown in SEQ ID NO :23 for Enterobacter aerogenes IFO 12010, in SEQ ID NO: 25 for Klebsiella planticola IFO 14939 and in SEQ ID NO: 27 for Serratia ficaria IAM 13540. Additionally, the deduced amino acid sequences are shown in SEQ ID NO: 24, 26 and 28, respectively. Because of the fact that the transformants harboring the plasmids containing these fragments exhibited the transphosphorylation activity, it was identified that these open reading frames were the objective acid phosphatase genes.

The nucleotide sequences and the deduced amino acid sequences were respectively compared with known sequences for homology. Data bases of EMBL and SWISS-PROT were used. As a result, it has been revealed that the genes illustrated in SEQ ID NO: 23, 25 and 27 in the Sequence Listing are new genes. It is assumed that the protein encoded by the gene derived from Enterobacter aerogenes IFO 12010 comprises 248 amino acid residues, the protein encoded by the gene derived from Klebsiella planticola IFO 14939 comprises 248 amino acid residues and the protein encoded by the gene derived from Serratia ficaria IAM 13540 comprises 244 amino acid residues. There is a possibility that these proteins may be precursor proteins like the acid phosphatases derived from Morganella morganii and Escherichia blattae.

The amino acid sequences deduced from the nucleotide sequences are shown in FIG. 12 in one-letter Symbols together with the deduced amino acid sequence of the acid phosphatase derived from Morganella morganii NCIMB 10466 obtained in Example 8, that of Escherichia blattae JCM 1650 obtained in Example 12 and the known amino acid sequence of the acid phosphatase of Providencia stuartii (EMBL Accession number X64820). Common amino acid residues among all of the amino acids sequences are indicated with asterisks under the sequences in FIG. 12 .

As shown in FIG. 12, the amino acid sequences of the acid phosphatases derived from six strains are highly homologous each other and 130 amino acid residues are common among all of the amino acid sequences. Thus, it is assumed that these acid phosphatases have similar functions.

Amplification of Activity by Expressing Gene of Acid Phosphatase Derived from Enterobacter aerogenes, Klebsiella planticola and Serratia ficaria

Escherichia coli JM109/pPRP100 constructed in Example 23 , Escherichia coli JM109/pENP110 , Escherichia coli JM109/pKLP110 and Escherichia coli JM109/pSEP110 constructed in Example 24 were inoculated to an L-medium (50 ml) containing 100 μg/ml of ampicillin and 1 mM of IPTG, and were cultivated at 37° C. for 16 hours. Microbial cells were harvested from these cultures by centrifugation, and they were washed once with physiological saline. The microbial cells were suspended in 100 mM potassium phosphate buffer (5 ml, pH 7.0), and they were disrupted by means of a ultrasonic treatment performed at 4° C. for 20 minutes. The treated solutions were centrifuged to remove an insoluble fraction, and thus cell-free extracts were prepared.

The transphosphorylation activities of the obtained cell-free extracts were measured while using controls of cell-free extracts prepared from Providencia stuartii ATCC 29851 , Enterobacter aerogenes IFO 12010 , Klebsiella planticola IFO 14939 , Serratia ficaria IAM 13450, and Escherichia coli JM109 transformed with the plasmid pUC118 in the same manner as described above. The results are shown in Table 16. The transphosphorylation activities were low in all of the wild type strains. The transphosphorylation activity was not detected in Escherichia coli JM109/pUC118. On the other hand, the transformants of Escherichia coli JM109 to which the acid phosphatase genes were introduced exhibited high transphosphorylation activities in comparison with wild type strains. According to the result, it has been demonstrated that each of the introduced DNA fragment allow Escherichia coli to express the acid phosphatase at a high level.

Production of a Mutant Acid Phosphatase Gene Having an Improved Temperature Stability

As described in Examples 20, 21 and 22, the E. blattae -derived mutant acid phosphatase gene-containing strain produced in Example 19 expressed the considerable amount of the acid phosphatase. In the production of 5′-inosinic acid from pyrophosphoric acid and inosine using this strain, 5′-inosinic acid was formed and accumulated in the high conversion yield. The optimum reaction temperature of this acid phosphatase was 35° C. However, when this reaction was conducted at a higher temperature, the reaction rate was increased, and the reaction was conducted upon increasing the nucleoside concentration of the phosphate acceptor in the reaction solution. Accordingly, it was expected that the nucleoside-5′-phosphate ester could be produced more efficiently for a shorter period of time. Thus, the temperature stability of the enzyme was improved upon introducing the mutation into the E. blattae -derived acid phosphatase gene cloned in Example 19 by the site specific mutation method using PCR.

Plasmid pEPI380 containing the gene encoding the E. blattae JCM1650-derived mutant acid phosphatase described in Example 19 was used, and the site specific mutation was introduced into this plasmid DNA by the genetic engineering method to produce the gene encoding the mutant acid phosphatase having the increased temperature stability. pEPI380 is a plasmid DNA obtained by binding a DNA fragment of 2.4 Kbp containing the gene encoding the mutant acid phosphatase derived from E. blattae JCM1650 and cleaved with restriction endonucleases ClaI and BamHI to pBluescript KS(+) (supplied by Stratagene) cleaved with ClaI and BamHI. The amino acid sequence of the mature protein anticipated from the base sequence of the gene encoding the acid phosphatase is presumed to be 11 amino acid resides shown in Table 12 in Example 19 in the sequence represented by SEQ ID NO: 8 in the Sequence Listing.

Oligonucleotides MUT300 (SEQ ID NO: 9 in the Sequence Listing), MUT400 (SEQ ID NO: 29 in the Sequence Listing) and MUT410 (SEQ ID NO: 30 in the Sequence Listing) having the sequences shown in the Sequence Table were synthesized by the phosphoamidite method using a DNA synthesizer (Model 394 supplied by Applied Biosystem).

A mutant gene encoding a mutant phosphatase in which the 104th glutamic acid residue (GAG) of a maturation protein was substituted with a glycine residue (GG*T*) was produced by the method with PCR as in Example 15 using pEPI380 described in Example 19 as a template and MUT300 and MUT410 as primers for introduction of mutation. This mutant gene-containing plasmid was designated pEPI410. Likewise, a mutant gene encoding a mutant phosphatase in which the 151st threonine residue (ACC) was substituted with an alanine residue (G*CC) was produced using pEPI380 as a template and oligonucleotides MUT300, MUT310 and MUT420 as primers for introduction of mutation. This mutant gene-containing plasmid was designated pEPI420.

A plasmid was produced from the transformant of E. coli JM109 having introduced therein plasmids pEPI410 and pEPI420 containing the mutant phosphatase gene by the alkali bacteriolysis method, the base sequence was determined, and it was identified that the desired base was substituted.

Each of E. coli JM109/pEPI410 and E. coli JM109/pEPI420 having introduced therein the mutant acid phosphatase gene as produced in this Example and E. coli JM109/pEPI380 described in Example 19 was inoculated in 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37° C. for 16 hours. The cells were collected from 50 ml of the culture solution of each of the strains, and washed once with a physiological saline solution. These cells were suspended in 5 ml of a 100-mM phosphate buffer (pH 7.0), and sonicated at 4° C. for 20 minutes to mill the cells. The thus-treated solution was centrifuged to remove insoluble fractions and prepare a cell-free extract.

The cell-free extract formed from each of the strains was warmed at temperatures ranging from 0° C. to 80° C. with a pH of 7.0 for 30 minutes. After the completion of the warming, the transphosphorylation was conducted under the standard reaction conditions of pH of 4.0 and 30° C. using the cell-free extracts treated at various temperatures, and the residual activity was measured. The results are shown in FIG. 13 . The mutant enzyme expressed in E. coli JM109/pEPI380 described in Example 19 was stable in the treatment at 40° C. for 30 minutes, but the decrease in the activity was observed at higher temperatures. By contrast, the new mutant enzyme expressed in E. coli JM109/pEPI410 and E. coli JM109/pEPI420 having introduced therein the new mutant enzyme gene as produced in this Example improved the temperature stability, and the decrease in the activity was not observed even through the treatment at 50° C. for 30 minutes. It was thus expected that when a nucleoside-5′-phosphate ester was produced using these strains at a high temperature, the productivity was further improved.

Production of 5′-Inosinic Acid and 5′-Guanylic Acid using a Mutant Acid Phosphatase Gene-Containing Strain Having an Improved Temperature Stability

Each of E. coli JM109/pEPI410 and E. coli JM109/pEPI420 having been introduced therein with the mutant acid phosphatase gene and E. coli JM109/pEPI380 described in Example 19 was inoculated in 50 ml of an L medium containing 100 μg/ml of ampicillin and 1 mM of IPTG, and incubated at 37° C. for 16 hours.

Pyrophosphoric acid (15 g/dl) and 8 g/dl of inosine or guanosine were dissolved in an acetate buffer (pH 4.0). To this was added E. coli JM109 strain having introduced therein each mutant acid phosphatase gene such that the concentration reached 100 mg/dl in terms of the dry cell weight. The reaction was conducted at 50° C. for 9 hours while maintaining the pH at 4.0, and the amount of 5′-inosinic acid or 5′-guanylic acid formed was measured. The results are shown in Table 17. The nucleoside phosphate ester formed was only a nucleoside-5′-phosphate ester, and the production of a nucleoside-2′-phosphate ester and a nucleoside-3′-phosphate ester as by-products was not observed at all. The reaction was also conducted at 30° C. for 12 hours using E. coli JM109/pEPI380 strain as a control. The results are also shown in Table 17.

As described in Example 21, the nucleoside-5′-phosphate ester was formed and accumulated efficiently with E. coli JM109/pEPI380. By contrast, when the reaction was conducted using E. coli JM109/pEPI410 and E. coli JM109/pEPI420 having been introduced therein with the new mutant acid phosphatase gene derived from E. blattae as produced in Example 26, 5′-inosinic acid or 5′-guanylic acid in the same amount was formed and accumulated for a shorter period of time. Thus, the nucleoside-5′-phosphate ester could be produced more efficiently. Especially when using E. coli JM109/pEPI420, not only was the reaction time shortened, but also were 5′-inosinic acid and 5′-guanylic acid accumulated in larger amounts, and quite a high productivity was shown.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

This application is based on Japanese Patent Application No. 311103/1996, filed Nov. 21, 1996, and Japanese Patent Application No. 161674/1997, filed Jun. 18, 1997, both of which are incorporated herein by reference in their entirety.

Ala Ile Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro Asp Leu Tyr Tyr

1 5 10 15

Leu Lys Asn Glu

ATG AAG AAG AAT ATT ATC GCC GGT TGT CTG TTC TCA CTG TTT TCC CTT 48

Met Lys Lys Asn Ile Ile Ala Gly Cys Leu Phe Ser Leu Phe Ser Leu

-20 -15 -10 -5

TCC GCG CTG GCC GCG ATC CCG GCG GGC AAC GAT GCC ACC ACC AAG CCG 96

Ser Ala Leu Ala Ala Ile Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

1 5 10

GAT TTA TAT TAT CTG AAA AAT GAA CAG GCT ATC GAC AGC CTG AAA CTG 144

Asp Leu Tyr Tyr Leu Lys Asn Glu Gln Ala Ile Asp Ser Leu Lys Leu

15 20 25

TTA CCG CCA CCG CCG GAA GTC GGC AGT ATT CAG TTT TTA AAT GAT CAG 192

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Gln Phe Leu Asn Asp Gln

30 35 40

GCA ATG TAT GAG AAA GGC CGT ATG CTG CGC AAT ACC GAG CGC GGA AAA 240

Ala Met Tyr Glu Lys Gly Arg Met Leu Arg Asn Thr Glu Arg Gly Lys

45 50 55 60

CAG GCA CAG GCA GAT GCT GAC CTG GCC GCA GGG GGT GTG GCA ACC GCA 288

Gln Ala Gln Ala Asp Ala Asp Leu Ala Ala Gly Gly Val Ala Thr Ala

65 70 75

TTT TCA GGG GCA TTC GGC TAT CCG ATA ACC GAA AAA GAC TCT CCG GAG 336

Phe Ser Gly Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ser Pro Glu

80 85 90

CTG TAT AAA CTG CTG ACC AAT ATG ATT GAG GAT GCC GGT GAT CTT GCC 384

Leu Tyr Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

95 100 105

ACC CGC TCC GCC AAA GAA CAT TAC ATG CGC ATC CGG CCG TTT GCG TTT 432

Thr Arg Ser Ala Lys Glu His Tyr Met Arg Ile Arg Pro Phe Ala Phe

110 115 120

TAC GGC ACA GAA ACC TGT AAT ACC AAA GAT CAG AAA AAA CTC TCC ACC 480

Tyr Gly Thr Glu Thr Cys Asn Thr Lys Asp Gln Lys Lys Leu Ser Thr

125 130 135 140

AAC GGA TCT TAC CCG TCA GGT CAT ACG TCT ATC GGC TGG GCA ACC GCA 528

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

145 150 155

CTG GTG CTG GCG GAA GTG AAC CCG GCA AAT CAG GAT GCG ATT CTG GAA 576

Leu Val Leu Ala Glu Val Asn Pro Ala Asn Gln Asp Ala Ile Leu Glu

160 165 170

CGG GGT TAT CAG CTC GGA CAG AGC CGG GTG ATT TGC GGC TAT CAC TGG 624

Arg Gly Tyr Gln Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

175 180 185

CAG AGT GAT GTG GAT GCC GCG CGG ATT GTC GGT TCA GCC GCT GTC GCG 672

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Ala Val Ala

190 195 200

ACA TTA CAT TCC GAT CCG GCA TTT CAG GCG CAG TTA GCG AAA GCC AAA 720

Thr Leu His Ser Asp Pro Ala Phe Gln Ala Gln Leu Ala Lys Ala Lys

205 210 215 220

CAG GAA TTT GCA CAA AAA TCA CAG AAA TAA 750

Gln Glu Phe Ala Gln Lys Ser Gln Lys

Met Lys Lys Asn Ile Ile Ala Gly Cys Leu Phe Ser Leu Phe Ser Leu

-20 -15 -10 -5

Ser Ala Leu Ala Ala Ile Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

1 5 10

Asp Leu Tyr Tyr Leu Lys Asn Glu Gln Ala Ile Asp Ser Leu Lys Leu

15 20 25

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Gln Phe Leu Asn Asp Gln

30 35 40

Ala Met Tyr Glu Lys Gly Arg Met Leu Arg Asn Thr Glu Arg Gly Lys

45 50 55 60

Gln Ala Gln Ala Asp Ala Asp Leu Ala Ala Gly Gly Val Ala Thr Ala

65 70 75

Phe Ser Gly Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ser Pro Glu

80 85 90

Leu Tyr Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

95 100 105

Thr Arg Ser Ala Lys Glu His Tyr Met Arg Ile Arg Pro Phe Ala Phe

110 115 120

Tyr Gly Thr Glu Thr Cys Asn Thr Lys Asp Gln Lys Lys Leu Ser Thr

125 130 135 140

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

145 150 155

Leu Val Leu Ala Glu Val Asn Pro Ala Asn Gln Asp Ala Ile Leu Glu

160 165 170

Arg Gly Tyr Gln Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

175 180 185

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Ala Val Ala

190 195 200

Thr Leu His Ser Asp Pro Ala Phe Gln Ala Gln Leu Ala Lys Ala Lys

205 210 215 220

Gln Glu Phe Ala Gln Lys Ser Gln Lys

Ala Ile Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro Asp Leu Tyr Tyr

1 5 10 15

Leu Lys Asn Glu Gln Ala Ile Asp Ser Leu Lys Leu Leu Pro Pro Pro

20 25 30

Pro Glu Val Gly Ser Ile Gln Phe Leu Asn Asp Gln Ala Met Tyr Glu

35 40 45

Lys Gly Arg Met Leu Arg Asn Thr Glu Arg Gly Lys Gln Ala Gln Ala

50 55 60

Asp Ala Asp Leu Ala Ala Gly Gly Val Ala Thr Ala Phe Ser Gly Ala

65 70 75 80

Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ser Pro Glu Leu Tyr Lys Leu

85 90 95

Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala Thr Arg Ser Ala

100 105 110

Lys Glu His Tyr Met Arg Ile Arg Pro Phe Ala Phe Tyr Gly Thr Glu

115 120 125

Thr Cys Asn Thr Lys Asp Gln Lys Lys Leu Ser Thr Asn Gly Ser Tyr

130 135 140

Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala Leu Val Leu Ala

145 150 155 160

Glu Val Asn Pro Ala Asn Gln Asp Ala Ile Leu Glu Arg Gly Tyr Gln

165 170 175

Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp Gln Ser Asp Val

180 185 190

Asp Ala Ala Arg Ile Val Gly Ser Ala Ala Val Ala Thr Leu His Ser

195 200 205

Asp Pro Ala Phe Gln Ala Gln Leu Ala Lys Ala Lys Gln Glu Phe Ala

210 215 220

Gln Lys Ser Gln Lys

225 229

Leu Ala Leu Val Ala Thr Gly Asn Asp Thr Thr Thr Lys Pro Asp Leu

1 5 10 15

ATG AAA AAA CGT GTT CTG GCA GTT TGT TTT GCC GCA TTG TTC TCT TCT 48

Met Lys Lys Arg Val Leu Ala Val Cys Phe Ala Ala Leu Phe Ser Ser

-18 -15 -10 -5

CAG GCC CTG GCG CTG GTC GCT ACC GGC AAC GAC ACT ACC ACG AAA CCG 96

Gln Ala Leu Ala Leu Val Ala Thr Gly Asn Asp Thr Thr Thr Lys Pro

1 5 10

GAT CTC TAC TAC CTC AAG AAC AGT GAA GCC ATT AAC AGC CTG GCG CTG 144

Asp Leu Tyr Tyr Leu Lys Asn Ser Glu Ala Ile Asn Ser Leu Ala Leu

15 20 25 30

TTG CCG CCA CCA CCG GCG GTG GGC TCC ATT GCG TTT CTC AAC GAT CAG 192

Leu Pro Pro Pro Pro Ala Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

35 40 45

GCC ATG TAT GAA CAG GGG CGC CTG CTG CGC AAC ACC GAA CGC GGT AAG 240

Ala Met Tyr Glu Gln Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys

50 55 60

CTG GCG GCG GAA GAT GCA AAC CTG AGC AGT GGC GGG GTG GCG AAT GCT 288

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ser Gly Gly Val Ala Asn Ala

65 70 75

TTC TCC GGC GCG TTT GGT AGC CCG ATC ACC GAA AAA GAC GCC CCG GCG 336

Phe Ser Gly Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Ala

80 85 90

CTG CAT AAA TTA CTG ACC AAT ATG ATT GAG GAC GCC GGG GAT CTG GCG 384

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

95 100 105 110

ACC CGC AGC GCG AAA GAT CAC TAT ATG CGC ATT CGT CCG TTC GCG TTT 432

Thr Arg Ser Ala Lys Asp His Tyr Met Arg Ile Arg Pro Phe Ala Phe

115 120 125

TAT GGG GTC TCT ACC TGT AAT ACC ACC GAG CAG GAC AAA CTG TCC AAA 480

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

130 135 140

AAT GGC TCT TAT CCG TCC GGG CAT ACC TCT ATC GGC TGG GCT ACT GCG 528

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

145 150 155

CTG GTG CTG GCA GAG ATC AAC CCT CAG CGC CAG AAC GAG ATC CTG AAA 576

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

160 165 170

CGC GGT TAT GAG CTG GGC CAG AGC CGG GTG ATT TGC GGC TAC CAC TGG 624

Arg Gly Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

175 180 185 190

CAG AGT GAT GTG GAT GCC GCG CGG GTA GTG GGA TCT GCC GTT GTG GCG 672

Gln Ser Asp Val Asp Ala Ala Arg Val Val Gly Ser Ala Val Val Ala

195 200 205

ACC CTG CAT ACC AAC CCG GCG TTC CAG CAG CAG TTG CAG AAA GCG AAG 720

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

210 215 220

GCC GAA TTC GCC CAG CAT CAG AAG AAA TAA 750

Ala Glu Phe Ala Gln His Gln Lys Lys

225 230

Met Lys Lys Arg Val Leu Ala Val Cys Phe Ala Ala Leu Phe Ser Ser

-18 -15 -10 -5

Gln Ala Leu Ala Leu Val Ala Thr Gly Asn Asp Thr Thr Thr Lys Pro

1 5 10

Asp Leu Tyr Tyr Leu Lys Asn Ser Glu Ala Ile Asn Ser Leu Ala Leu

15 20 25 30

Leu Pro Pro Pro Pro Ala Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

35 40 45

Ala Met Tyr Glu Gln Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys

50 55 60

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ser Gly Gly Val Ala Asn Ala

65 70 75

Phe Ser Gly Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Ala

80 85 90

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

95 100 105 110

Thr Arg Ser Ala Lys Asp His Tyr Met Arg Ile Arg Pro Phe Ala Phe

115 120 125

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

130 135 140

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

145 150 155

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

160 165 170

Arg Gly Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

175 180 185 190

Gln Ser Asp Val Asp Ala Ala Arg Val Val Gly Ser Ala Val Val Ala

195 200 205

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

210 215 220

Ala Glu Phe Ala Gln His Gln Lys Lys

225 230

Leu Ala Leu Val Ala Thr Gly Asn Asp Thr Thr Thr Lys Pro Asp Leu

1 5 10 15

Tyr Tyr Leu Lys Asn Ser Glu Ala Ile Asn Ser Leu Ala Leu Leu Pro

20 25 30

Pro Pro Pro Ala Val Gly Ser Ile Ala Phe Leu Asn Asp Gln Ala Met

35 40 45

Tyr Glu Gln Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys Leu Ala

50 55 60

Ala Glu Asp Ala Asn Leu Ser Ser Gly Gly Val Ala Asn Ala Phe Ser

65 70 75 80

Gly Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Ala Leu His

85 90 95

Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala Thr Arg

100 105 110

Ser Ala Lys Asp His Tyr Met Arg Ile Arg Pro Phe Ala Phe Tyr Gly

115 120 125

Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys Asn Gly

130 135 140

Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala Leu Val

145 150 155 160

Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys Arg Gly

165 170 175

Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp Gln Ser

180 185 190

Asp Val Asp Ala Ala Arg Val Val Gly Ser Ala Val Val Ala Thr Leu

195 200 205

His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys Ala Glu

210 215 220

Phe Ala Gln His Gln Lys Lys

225 230

CCTCGAGGTC GACGGTATCG 20

ATTCGCCACA TCGCCACTGC T 21

TAGCCCAGCC GGTAGAGGTA TG 22

TGCATCTGCC TGCGCCTGCT TAC 23

AACGCGCCGT AGAAAGCATT 20

GTCCTGGTCT TTGGTATTAC A 21

CACATCGCCA GCGGCCAGGT CTGCAT 26

GCATATAGTG TTCTTTCGCG C 21

ATTACAGGTT TCGACCCCAT AA 22

TGATGCATGT CCGGGCTGTC TTTTT 25

CTGGATCCTG TGGCTATCAT CACCT 25

CTGGATCCGA CGCGATTTTA CCATA 25

ATG AAA AAA CTA TTA GCA GTA TTC TGC GCA GGG GCT TTT GTT TCA ACC 48

Met Lys Lys Leu Leu Ala Val Phe Cys Ala Gly Ala Phe Val Ser Thr

1 5 10 15

AGT GTA TTT GCG GCG ATC CCT CCC GGC AAT GAT GTG ACA ACT AAA CCC 96

Ser Val Phe Ala Ala Ile Pro Pro Gly Asn Asp Val Thr Thr Lys Pro

20 25 30

GAT CTT TAT TAT TTA AAA AAC TCA CAG GCT ATT GAT AGT TTA GCG TTA 144

Asp Leu Tyr Tyr Leu Lys Asn Ser Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

TTG CCG CCA CCA CCT GAA GTG GGC AGT ATC TTA TTT TTA AAC GAC CAA 192

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Leu Phe Leu Asn Asp Gln

50 55 60

GCG ATG TAT GAA AAA GGC CGT TTA TTG CGA AAT ACT GAG CGT GGA GAA 240

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Glu

65 70 75 80

CAA GCC GCT AAG GAT GCT GAT CTG GCT GCG GGC GGT GTT GCG AAC GCA 288

Gln Ala Ala Lys Asp Ala Asp Leu Ala Ala Gly Gly Val Ala Asn Ala

85 90 95

TTT TCT GAA GCT TTT GGT TAT CCC ATT ACC GAA AAG GAT GCG CCT GAA 336

Phe Ser Glu Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ala Pro Glu

100 105 110

ATT CAT AAA TTG CTG ACG AAT ATG ATT GAA GAT GCG GGG GAT TTA GCA 384

Ile His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

ACT CGC TCA GCC AAA GAG AAA TAC ATG CGC ATT CGT CCA TTT GCG TTC 432

Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

TAC GGT GTT GCT ACC TGT AAC ACG AAA GAT CAG GAC AAA TTA TCT AAG 480

Tyr Gly Val Ala Thr Cys Asn Thr Lys Asp Gln Asp Lys Leu Ser Lys

145 150 155 160

AAT GGC TCT TAT CCT TCT GGA CAC ACC GCA ATT GGC TGG GCA TCT GCA 528

Asn Gly Ser Tyr Pro Ser Gly His Thr Ala Ile Gly Trp Ala Ser Ala

165 170 175

CTC GTA TTG TCA GAA ATT AAC CCA GAA AAC CAA GAT AAA ATT TTA AAA 576

Leu Val Leu Ser Glu Ile Asn Pro Glu Asn Gln Asp Lys Ile Leu Lys

180 185 190

CGT GGT TAT GAA CTT GGC CAA AGC CGA GTC ATC TGT GGT TAC CAT TGG 624

Arg Gly Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

CAA AGT GAT GTT GAT GCA GCT CGT ATC GTT GCA TCG GGT GCG GTA GCA 672

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Ala Ser Gly Ala Val Ala

210 215 220

ACT TTA CAC TCC AAC CCT GAA TTC CAA AAA CAG TTA CAA AAA GCC AAA 720

Thr Leu His Ser Asn Pro Glu Phe Gln Lys Gln Leu Gln Lys Ala Lys

225 230 235 240

GAC GAA TTT GCT AAA CTG AAA AAA TAG 747

Asp Glu Phe Ala Lys Leu Lys Lys

Met Lys Lys Leu Leu Ala Val Phe Cys Ala Gly Ala Phe Val Ser Thr

1 5 10 15

Ser Val Phe Ala Ala Ile Pro Pro Gly Asn Asp Val Thr Thr Lys Pro

20 25 30

Asp Leu Tyr Tyr Leu Lys Asn Ser Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Leu Phe Leu Asn Asp Gln

50 55 60

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Glu

65 70 75 80

Gln Ala Ala Lys Asp Ala Asp Leu Ala Ala Gly Gly Val Ala Asn Ala

85 90 95

Phe Ser Glu Ala Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ala Pro Glu

100 105 110

Ile His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

Tyr Gly Val Ala Thr Cys Asn Thr Lys Asp Gln Asp Lys Leu Ser Lys

145 150 155 160

Asn Gly Ser Tyr Pro Ser Gly His Thr Ala Ile Gly Trp Ala Ser Ala

165 170 175

Leu Val Leu Ser Glu Ile Asn Pro Glu Asn Gln Asp Lys Ile Leu Lys

180 185 190

Arg Gly Tyr Glu Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Ala Ser Gly Ala Val Ala

210 215 220

Thr Leu His Ser Asn Pro Glu Phe Gln Lys Gln Leu Gln Lys Ala Lys

225 230 235 240

Asp Glu Phe Ala Lys Leu Lys Lys

ATG AAA AAG CGC GTT CTC GCC CTC TGC CTC GCC AGC CTG TTT TCC GTT 48

Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val

1 5 10 15

AAC GCT TTC GCG CTG GTC CCT GCC GGC AAT GAT GCA ACC ACC AAA CCG 96

Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

20 25 30

GAT CTC TAT TAT CTG AAA AAT GCA CAG GCC ATC GAT AGT CTG GCG CTG 144

Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

TTG CCG CCG CCG CCG GAA GTT GGC AGC ATC GCA TTT TTA AAC GAT CAG 192

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

50 55 60

GCG ATG TAT GAG AAA GGA CGG CTG TTG CGC AAT ACC GAA CGT GGC AAG 240

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys

65 70 75 80

CTG GCG GCT GAA GAT GCT AAC CTG AGC GCC GGC GGC GTC GCG AAT GCC 288

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ala Gly Gly Val Ala Asn Ala

85 90 95

TTC TCC AGC GCT TTT GGT TCG CCC ATC ACC GAA AAA GAC GCG CCG CAG 336

Phe Ser Ser Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Gln

100 105 110

TTA CAT AAG CTG CTG ACA AAT ATG ATT GAG GAT GCC GGC GAT CTG GCC 384

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

ACC CGC AGC GCG AAA GAG AAA TAT ATG CGC ATT CGC CCG TTT GCG TTC 432

Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

TAC GGC GTT TCA ACC TGT AAC ACT ACC GAG CAG GAC AAG CTG TCG AAA 480

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

145 150 155 160

AAC GGA TCT TAC CCT TCC GGC CAT ACC TCT ATC GGT TGG GCA ACC GCG 528

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

165 170 175

CTG GTA CTG GCG GAG ATC AAT CCG CAG CGG CAA AAC GAA ATT CTC AAA 576

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

180 185 190

CGC GGC TAT GAA TTG GGC GAA AGC CGG GTT ATC TGC GGC TAT CAT TGG 624

Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

CAG AGC GAT GTC GAT GCG GCG CGG ATA GTC GGC TCG GCG GTG GTG GCG 672

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala

210 215 220

ACC CTG CAT ACC AAC CCG GCC TTC CAA CAG CAG TTG CAG AAA GCA AAG 720

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

225 230 235 240

GAT GAA TTC GCC AAA ACG CAG AAG TAA 747

Asp Glu Phe Ala Lys Thr Gln Lys

Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val

1 5 10 15

Asn Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

20 25 30

Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

50 55 60

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Asn Thr Glu Arg Gly Lys

65 70 75 80

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ala Gly Gly Val Ala Asn Ala

85 90 95

Phe Ser Ser Ala Phe Gly Ser Pro Ile Thr Glu Lys Asp Ala Pro Gln

100 105 110

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

Thr Arg Ser Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

145 150 155 160

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

165 170 175

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

180 185 190

Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala

210 215 220

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

225 230 235 240

Asp Glu Phe Ala Lys Thr Gln Lys

ATG AAA AAG CGT GTA CTC GCC CTT TGC CTT GCC AGC CTC TTT TCA GTT 48

Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val

1 5 10 15

AGC GCC TTT GCG CTG GTT CCC GCC GGC AAT GAT GCC ACC ACC AAG CCC 96

Ser Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

20 25 30

GAT CTC TAC TAT CTG AAA AAT GCC CAG GCC ATT GAC AGC CTG GCG CTG 144

Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

TTG CCA CCG CCG CCG GAA GTG GGC AGC ATT GCG TTT TTA AAC GAT CAG 192

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

50 55 60

GCG ATG TAT GAG AAA GGC CGT CTG CTG CGC GCC ACC GCC CGC GGC AAG 240

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Ala Thr Ala Arg Gly Lys

65 70 75 80

TTG GCG GCA GAA GAT GCC AAC CTG AGC GCG GGT GGC GTG GCC AAC GCC 288

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ala Gly Gly Val Ala Asn Ala

85 90 95

TTC TCC GCA GCA TTC GGC TCC CCG ATC AGC GAA AAA GAC GCC CCG GCG 336

Phe Ser Ala Ala Phe Gly Ser Pro Ile Ser Glu Lys Asp Ala Pro Ala

100 105 110

CTG CAC AAA CTG CTC ACC AAC ATG ATT GAA GAC GCG GGC GAT CTG GCG 384

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

ACC CGA GGC GCG AAA GAG AAG TAT ATG CGT ATT CGT CCG TTT GCC TTC 432

Thr Arg Gly Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

TAC GGC GTG TCC ACC TGC AAT ACC ACC GAA CAG GAT AAG CTG TCG AAA 480

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

145 150 155 160

AAC GGC TCC TAC CCT TCC GGA CAC ACC TCT ATC GGC TGG GCG ACC GCC 528

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

165 170 175

CTG GTG CTG GCC GAA ATC AAC CCG CAG CGC CAG AAT GAG ATT CTC AAG 576

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

180 185 190

CGC GGC TAT GAG CTC GGT GAA AGT CGG GTG ATC TGC GGT TAC CAC TGG 624

Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

CAG AGC GAT GTT GAC GCC GCG CGG ATT GTC GGC TCG GCG GTG GTT GCA 672

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala

210 215 220

ACC CTG CAT ACC AAT CCG GCC TTC CAG CAG CAG CTG CAA AAA GCC AAA 720

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

225 230 235 240

GAC GAG TTT GCG AAA CAG CAG AAA TAG 747

Asp Glu Phe Ala Lys Gln Gln Lys

Met Lys Lys Arg Val Leu Ala Leu Cys Leu Ala Ser Leu Phe Ser Val

1 5 10 15

Ser Ala Phe Ala Leu Val Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro

20 25 30

Asp Leu Tyr Tyr Leu Lys Asn Ala Gln Ala Ile Asp Ser Leu Ala Leu

35 40 45

Leu Pro Pro Pro Pro Glu Val Gly Ser Ile Ala Phe Leu Asn Asp Gln

50 55 60

Ala Met Tyr Glu Lys Gly Arg Leu Leu Arg Ala Thr Ala Arg Gly Lys

65 70 75 80

Leu Ala Ala Glu Asp Ala Asn Leu Ser Ala Gly Gly Val Ala Asn Ala

85 90 95

Phe Ser Ala Ala Phe Gly Ser Pro Ile Ser Glu Lys Asp Ala Pro Ala

100 105 110

Leu His Lys Leu Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala

115 120 125

Thr Arg Gly Ala Lys Glu Lys Tyr Met Arg Ile Arg Pro Phe Ala Phe

130 135 140

Tyr Gly Val Ser Thr Cys Asn Thr Thr Glu Gln Asp Lys Leu Ser Lys

145 150 155 160

Asn Gly Ser Tyr Pro Ser Gly His Thr Ser Ile Gly Trp Ala Thr Ala

165 170 175

Leu Val Leu Ala Glu Ile Asn Pro Gln Arg Gln Asn Glu Ile Leu Lys

180 185 190

Arg Gly Tyr Glu Leu Gly Glu Ser Arg Val Ile Cys Gly Tyr His Trp

195 200 205

Gln Ser Asp Val Asp Ala Ala Arg Ile Val Gly Ser Ala Val Val Ala

210 215 220

Thr Leu His Thr Asn Pro Ala Phe Gln Gln Gln Leu Gln Lys Ala Lys

225 230 235 240

Asp Glu Phe Ala Lys Gln Gln Lys

ATG AAA AAA ATA TTA TTA GCC ACA TTA AGC TGC GCC GCG TTG ACG CAG 48

Met Lys Lys Ile Leu Leu Ala Thr Leu Ser Cys Ala Ala Leu Thr Gln

1 5 10 15

TTT TCC TTT GCC GCC AAA GAT GTC ACT ACC CAC CCT GAG GTT TAT TTT 96

Phe Ser Phe Ala Ala Lys Asp Val Thr Thr His Pro Glu Val Tyr Phe

20 25 30

CTG CAA GAA TCA CAG TCC ATC GAC AGC CTG GCA CTA TTG CCG CCG CCG 144

Leu Gln Glu Ser Gln Ser Ile Asp Ser Leu Ala Leu Leu Pro Pro Pro

35 40 45

CCG GCG ATG GAC AGC ATT GAT TTC CTG AAT GAC AAA GCG CAA TAC GAC 192

Pro Ala Met Asp Ser Ile Asp Phe Leu Asn Asp Lys Ala Gln Tyr Asp

50 55 60

GCC GGG AAA ATA GTG CGC AAT ACT CCG CGT GGC AAG CAG GCT TAT GAT 240

Ala Gly Lys Ile Val Arg Asn Thr Pro Arg Gly Lys Gln Ala Tyr Asp

65 70 75 80

GAC GCC CAC GTT GCC GGG GAC GGC GTT GCC GCC GCA TTT TCC AAC GCC 288

Asp Ala His Val Ala Gly Asp Gly Val Ala Ala Ala Phe Ser Asn Ala

85 90 95

TTC GGC CTA GAA ATA GCC CAA CGG AAA ACG CCG GAG CTG TTT AAG CTG 336

Phe Gly Leu Glu Ile Ala Gln Arg Lys Thr Pro Glu Leu Phe Lys Leu

100 105 110

GTG ATG AAA ATG CGT GAA GAC GCC GGC GAT TTG GCG ACC CGC AGC GCC 384

Val Met Lys Met Arg Glu Asp Ala Gly Asp Leu Ala Thr Arg Ser Ala

115 120 125

AAA AAT CAC TAT ATG CGC ATT CGC CCC TTT GCG TTT TAT AAC GAA GCG 432

Lys Asn His Tyr Met Arg Ile Arg Pro Phe Ala Phe Tyr Asn Glu Ala

130 135 140

ACC TGC CGA CCG GAC GAA GAA AGC ACC CTG TCG AAG AAC GGT TCT TAC 480

Thr Cys Arg Pro Asp Glu Glu Ser Thr Leu Ser Lys Asn Gly Ser Tyr

145 150 155 160

CCT TCC GGC CAT ACC ACC ATC GGC TGG GCG ACC GCG CTG GTG CTG GCT 528

Pro Ser Gly His Thr Thr Ile Gly Trp Ala Thr Ala Leu Val Leu Ala

165 170 175

GAA ATC AAC CCC GCC AGG CAG GGT GAA ATC CTG CAG CGC GGC TAT GAT 576

Glu Ile Asn Pro Ala Arg Gln Gly Glu Ile Leu Gln Arg Gly Tyr Asp

180 185 190

ATG GGC CAA AGC CGG GTT ATC TGC GGT TAT CAC TGG CAA AGC GAC GTG 624

Met Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp Gln Ser Asp Val

195 200 205

ACT GCG GCG CGC ATG GCG GCG TCG GCC ATG GTG GCG CGT TTG CAT GCC 672

Thr Ala Ala Arg Met Ala Ala Ser Ala Met Val Ala Arg Leu His Ala

210 215 220

GAA CCC ACC TTC GCC GCC CAG CTG CAA AAG GCC AAA GAC GAA TTC AAC 720

Glu Pro Thr Phe Ala Ala Gln Leu Gln Lys Ala Lys Asp Glu Phe Asn

225 230 235 240

GGC CTG AAA AAG TAA 735

Gly Leu Lys Lys

Met Lys Lys Ile Leu Leu Ala Thr Leu Ser Cys Ala Ala Leu Thr Gln

1 5 10 15

Phe Ser Phe Ala Ala Lys Asp Val Thr Thr His Pro Glu Val Tyr Phe

20 25 30

Leu Gln Glu Ser Gln Ser Ile Asp Ser Leu Ala Leu Leu Pro Pro Pro

35 40 45

Pro Ala Met Asp Ser Ile Asp Phe Leu Asn Asp Lys Ala Gln Tyr Asp

50 55 60

Ala Gly Lys Ile Val Arg Asn Thr Pro Arg Gly Lys Gln Ala Tyr Asp

65 70 75 80

Asp Ala His Val Ala Gly Asp Gly Val Ala Ala Ala Phe Ser Asn Ala

85 90 95

Phe Gly Leu Glu Ile Ala Gln Arg Lys Thr Pro Glu Leu Phe Lys Leu

100 105 110

Val Met Lys Met Arg Glu Asp Ala Gly Asp Leu Ala Thr Arg Ser Ala

115 120 125

Lys Asn His Tyr Met Arg Ile Arg Pro Phe Ala Phe Tyr Asn Glu Ala

130 135 140

Thr Cys Arg Pro Asp Glu Glu Ser Thr Leu Ser Lys Asn Gly Ser Tyr

145 150 155 160

Pro Ser Gly His Thr Thr Ile Gly Trp Ala Thr Ala Leu Val Leu Ala

165 170 175

Glu Ile Asn Pro Ala Arg Gln Gly Glu Ile Leu Gln Arg Gly Tyr Asp

180 185 190

Met Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp Gln Ser Asp Val

195 200 205

Thr Ala Ala Arg Met Ala Ala Ser Ala Met Val Ala Arg Leu His Ala

210 215 220

Glu Pro Thr Phe Ala Ala Gln Leu Gln Lys Ala Lys Asp Glu Phe Asn

225 230 235 240

Gly Leu Lys Lys

CCCGGCGTCA CCAATCATAT T 21

GCCGGTAGAG GCATGCCCGG A 21