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Applied and Environmental Microbiology, July 2000, p. 2811-2816, Vol. 66, No. 7
Applied Microbiology Laboratory, Fermentation
and Biotechnology Laboratories, Ajinomoto Co., Inc., Kawasaki-ku,
Kawasaki-shi 210-8681,1 and
Biotechnology Research Center, Toyama Prefectural University,
5180 Kurokawa, Kosugi, Toyama 939-0398,2
Japan
Received 8 February 2000/Accepted 24 April 2000
A novel nucleoside phosphorylation process using the food additive
pyrophosphate as the phosphate source was investigated. The
Morganella morganii gene encoding a selective nucleoside
pyrophosphate phosphotransferase was cloned. It was identical to the
M. morganii PhoC acid phosphatase gene. Sequential in vitro
random mutagenesis was performed on the gene by error-prone PCR to
construct a mutant library. The mutant library was introduced into
Escherichia coli, and the transformants were screened for
the production of 5'-IMP. One mutated acid phosphatase with an
increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of
5'-IMP per liter (192 mM) was synthesized from inosine in an 88% molar
yield. This improvement was achieved with two mutations, Gly to Asp at
position 92 and Ile to Thr at position 171. A decreased
Km value for inosine was responsible for the
increased productivity.
Nucleotides are often used as food
additives and as pharmaceutical intermediates. Among them, 5'-IMP and
5'-GMP are important, because they have a characteristic taste and are
used as flavor potentiators in various foods. Purine nucleosides such
as inosine (7, 9) and guanosine (8) can be
produced efficiently by fermentation, and phosphorylation of
nucleosides is a very efficient process for the large-scale production
of 5' nucleotides.
At present, there are two main phosphorylation methods. One is a
chemical phosphorylation process that uses phosphoryl chloride (POCl3) (22), and the other is an enzymatic
phosphorylation process that uses inosine kinase of Escherichia
coli (11, 12). The chemical phosphorylation process is
relatively complex, because it needs two reactors, for the fermentation
and chemical reactions. The enzymatic phosphorylation process is
simpler, because the enzymatic reaction can be carried out in the same
reactor as the fermentation reaction. The inosine kinase reaction,
however, requires ATP, and the ATP needs to be regenerated by resting
cells of Corynebacterium ammoniagenes, which are used for
the fermentative production of inosine. Therefore, applications of the
enzymatic phosphorylation process are limited. Alternatively, an enzyme
that catalyzes the synthesis of nucleotides by transfer of phosphate
groups from low-energy phosphate esters to nucleosides was described by
Brawerman and Chargaff (3) and Mitsugi and coworkers
(10).
Prompted by these findings, we have investigated a novel nucleoside
phosphorylation reaction using the food additive pyrophosphate (PPi), as shown in the following equation (9,
10): nucleoside + PPi In this paper we describe the cloning of the phosphotransferase gene
from M. morganii NCIMB10466, further optimization of the
reaction conditions, and improvement of enzyme activity by random mutation.
Strains, plasmids, and culture conditions.
M. morganii
NCIMB10466, which was previously selected as the strain producing the
highest level of 5' nucleotide using PPi as the phosphate
source (1), was used as the DNA donor. E. coli
JM109 (21) was used as the host strain for DNA manipulation and expression. Plasmids pUC18 and pUC19 (20) (Takara Shuzo, Kyoto, Japan) were used as vectors for E. coli.
Luria-Bertani (LB) medium (15) was used for the culturing of
M. morganii and E. coli. These microorganisms
were grown aerobically at 37°C. For the selection of E. coli transformants, ampicillin (50 µg/ml) was added to the medium.
General DNA manipulations.
All basic recombinant DNA
procedures, such as isolation and purification of DNA, restriction
enzyme digestion, ligation of DNA, and transformation of E. coli, were performed as described by Sambrook et al.
(15).
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Phosphorylation of Nucleosides by the Mutated Acid
Phosphatase from Morganella morganii
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
nucleoside
5'-monophosphate + Pi acid
phosphatase/phosphotransferase (EC 3.1.3.2). We purified and
characterized a C5'-position selective pyrophosphate-nucleoside
phosphotransferase from a crude extract of Morganella
morganii NCIMB10466 (2). The purified enzyme exhibited
not only phosphotransferase activity but also phosphatase activity. On
the basis of a kinetic study, it appeared to be a phosphatase with
regioselective phosphotransferase activity. In order for the enzyme to
be useful, it would be necessary to suppress the dephosphorylation
reaction and increase the efficiency of the transphosphorylation reaction.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Enzyme assays. Phosphotransferase activity was assayed in a standard reaction mixture containing 100 µmol of sodium acetate buffer (pH 5.0), 40 µmol of inosine, 100 µmol of tetrasodium pyrophosphate, and the enzyme solution in a total volume of 1 ml. Kinetic constants for inosine in the transphosphorylation reaction were measured at pH 4.0 using 100 µmol of sodium acetate buffer (pH 4.0). The reaction mixture was incubated for 10 min at 30°C, and then the reaction was stopped by adding 0.2 ml of 2 N HCl. Quantitative determination of inosine and 5'-IMP was carried out by high-pressure liquid chromatography (HPLC) using a Cosmosil 5C18-MS column (4.6 by 150 mm; Nacalai Tesque Co., Kyoto, Japan) with detection at 245 nm. The mobile phase was 5 mM potassium phosphate buffer (pH 2.8)-methanol (95:5, [vol/vol]), and the flow rate was 1 ml/min. One unit of phosphotransferase activity was defined as the amount of enzyme that produced 1 µmol of 5'-IMP per min under the assay conditions.
5'-Nucleotidase activity was assayed in a standard reaction mixture containing 100 µmol of sodium acetate buffer (pH 4.0), 10 µmol of 5'-IMP, and the enzyme solution in a total volume of 1 ml. The reaction mixture was incubated for 5 min at 30°C. The reaction was stopped by adding 0.2 ml of 2 N HCl, and then released inosine was measured by HPLC. One unit of 5'-nucleotidase activity was defined as the amount of enzyme that produced 1 µmol of inosine per min under the assay conditions. Phosphatase activity was assayed by monitoring the rate of hydrolysis of p-nitrophenyl phosphate (p-NPP). The reaction mixture contained 100 µmol of morpholineethanesulfonic acid (MES)-NaOH buffer (pH 6.0), 10 µmol of p-NPP, and the enzyme solution in a total volume of 1 ml. The reaction mixture was incubated for 1 min at 30°C, and then the reaction was stopped by adding 0.2 ml of 2 N KOH. The release of p-nitrophenol was measured at 410 nm. One unit of phosphatase activity was defined as the amount of enzyme that produced 1 µmol of p-nitrophenol per min under the assay conditions. Kinetic constants for inosine in the transphosphorylation reaction and for 5'-IMP in the dephosphorylation reaction were measured at pH 4.0, at which 5'-IMP synthesis was carried out. Kinetic constants for p-NPP in the dephosphorylation reaction were measured at pH 6.0, at which the enzyme has optimal activity. The initial velocities were determined under the assay conditions described above, and the steady-state kinetic constants were calculated by using a Lineweaver-Burk plot. As the solubility of inosine was limited, kinetic constants for inosine were determined with a substrate concentration ranging from 0.5 to 80 mM.Preparation of internal peptides of the phosphotransferase from M. morganii. The purified phosphotransferase from M. morganii (1 mg) (2) was digested with 15 µg of lysyl endopeptidase (Wako, Kyoto, Japan) in 1 ml of 15 mM Tris-HCl buffer (pH 9.0) at 30°C for 16 h. The reaction mixture was subsequently eluted by HPLC on a TSK gel octadecyl silane-80Ts column (4.5 by 150 mm; Tosoh, Tokyo, Japan) with a 20 to 70% linear gradient of CH3CN containing 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min, and each peptide fragment was collected. The amino acid sequences of the peptides and of the amino-terminal region were analyzed with an automated protein sequencer (Prosequencer 6625; Millipore Corp.).
Cloning and nucleotide sequencing of the phosphotransferase
gene.
An M. morganii chromosomal DNA library was
constructed by inserting partial Sau3AI-digested fragments
of 4 to 8 kb into the BamHI site of pUC18. E. coli JM109 transformants were grown on LB plates containing 50 µg of ampicillin per ml and 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for 16 h.
To visualize colonies of transformants showing acid phosphatase
activity, 2 ml of 0.1 M MES-NaOH buffer (pH 6.0) containing 4 mM
p-NPP was poured onto the surface of the plates. We then
screened for phosphotransferase-positive clones among the
phosphatase-positive candidates. Each candidate was cultured in 4 ml of
LB medium containing 50 µg of ampicillin per ml and 1 mM IPTG at
37°C for 16 h. Recombinant cells were harvested by centrifugation, and phosphotransferase activity was measured as described. Recombinants that produced 5'-IMP were selected and used for
further study.
In vitro random mutagenesis and screening of mutants. Random mutagenesis of the PhoC gene was performed by error-prone PCR according to the method of Cadwell and Joyce (4, 5). The mutagenic reaction mixtures contained 20 fmol of plasmid pMPI501 as a template, 30 pmol of M13 primer RV, 30 pmol of M13 primer M4 (Takara Shuzo), 50 mM KCl, 10 mM Tris-HCl buffer (pH 8.3), 7 mM MgCl2, 0.5 mM MnCl2, 0.2 mM each dATP and dGTP, 1 mM each dTTP and dCTP, and 5 U of Taq DNA polymerase (Pharmacia) in a total volume of 100 µl. PCR was carried out with programs of 30 cycles of 94°C for 1 min, 42°C for 2 min, and 72°C for 3 min. PCR products were purified by chloroform-isoamyl alcohol extraction and ethanol precipitation. A mixture of EcoRI-HindIII fragments including the mutagenized PhoC gene was cut out and religated into the pUC18 plasmid to generate a mutant library.
The constructed mutant library was transformed with E. coli JM109. Each transformant was isolated and cultivated in LB broth as described above. A phosphotransferase reaction was then carried out with each of the clones. The reaction mixture contained 20 g of inosine per liter (75 mM), 100 g of tetrasodium pyrophosphate decahydrate per liter (224 mM), 0.1 M sodium acetate buffer (pH 4.0), and approximately 50 mg (wet weight, harvested from 3 ml of culture broth) of E. coli JM109 transformants in 0.5 ml. After incubation for 2, 6, and 16 h at 30°C, the amount of 5'-IMP produced was measured. Candidates that produced a larger amount of 5'-IMP and showed lower nucleotidase activity than E. coli JM109 harboring wild-type phoC were selected. The second round of random mutagenesis was performed by the same procedure, using as a template a plasmid derived from an improved variant produced in the first round of mutagenesis and chosen as the parent for the next generation.Site-directed mutagenesis. To prepare a PhoC acid phosphatase mutant with the G92D mutation, the following mutagenic primer was synthesized: 5'-GCGGTTGCCACA(C to T)CCCCTGCG-3'. The target mutation was introduced into plasmid pMPI501 with the MUT1 primer (Takara Shuzo) and the mutagenic primer (for the first PCR) and with M13 primer RV and M13 primer M4 (for the first and second PCRs) via heteroduplex formation between the first two PCR products (6). We used an in vitro mutagenesis kit (Takara Shuzo). PCR was carried out with programs of 25 cycles of 94°C for 30 s, 55°C for 2 min, and 72°C for 3 min (for the first PCR) and 10 cycles under the same conditions as those used for the first PCR (for the second PCR). The single-stranded region of the heteroduplex was filled in by the second PCR, followed by double digestion with EcoRI and HindIII. The double-stranded DNA fragment carrying the mutation could, in theory, be selectively digested with both enzymes and cloned into the same restriction site of plasmid pUC18. The 1.2-kb EcoRI-HindIII fragment religated into pUC18 was designated pMPI502, and the mutation was confirmed by sequencing.
Synthesis of 5'-IMP by E. coli overproducing wild-type and mutated PhoC. Each E. coli JM109 transformant subcultured on LB agar containing 50 µg of ampicillin per ml was inoculated into 200 ml of LB medium containing 50 µg of ampicillin per ml and 1 mM IPTG in 500-ml flasks and cultured aerobically on a reciprocal shaker at 37°C for 20 h. The cells were harvested from the culture broth by centrifugation at 10,000 × g for 10 min. The harvested cells were washed with 10 mM potassium phosphate buffer (pH 7.0) and then suspended in the same buffer. Cell growth was estimated turbidimetrically by means of a dry-cell calibration curve for the absorbance at 610 nm: 0.24 mg of dry cell weight per ml was equivalent to 1.0 U of optical density at 610 nm. Approximately 200 mg (dry weight) of cells was obtained from 200 ml of culture broth. The standard reaction mixture for 5'-IMP synthesis contained various concentrations of inosine, 150 g of disodium hydrogen pyrophosphate per liter (200 mM), 1 mM sodium acetate buffer (pH 4.0), and 1 g (dry weight) of cells per liter in a 10-ml volume. The reaction was carried out at 30°C with moderate shaking and stopped by adding 1 ml of 2 N HCl. Synthesized 5'-IMP was calculated as IMP · 2Na · 7.5H2O (molecular weight, 527).
Enzyme purification. Each acid phosphatase was purified from harvested cells of E. coli JM109 transformants harboring wild-type or mutated phoC by ammonium sulfate fractionation and ion-exchange, hydrophobic, and gel filtration column chromatographies as described previously (2). The purity of the recovered samples was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14% polyacrylamide).
Nucleotide sequence accession number. The nucleotide sequence reported in this paper has been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB35805.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation of an M. morganii pyrophosphate-nucleoside phosphotransferase gene. For mutagenesis, an M. morganii gene encoding a pyrophosphate-nucleoside phosphotransferase was isolated by a shotgun cloning strategy. As the enzyme appeared to be a phosphatase (2), phosphatase-positive clones were selected, and their phosphotransferase activity was examined. Of about 10,000 transformants examined, 42 were phosphatase positive. Among these 42 candidates, 3 transformants showed phosphotransferase activity. These three may overlap, because a 1.2-kb EcoRI-HindIII fragment was contained in the plasmids rescued from each candidate.
The results of subcloning showed that phosphotransferase activity was retained on a 1.2-kb EcoRI-HindIII fragment. The 1.2-kb fragment was subcloned into pUC18 and designated pMPI501. The nucleotide sequence of the fragment (given in the database) revealed an open reading frame (ORF) encoding the 747-bp phosphotransferase gene (Fig. 1). The amino-terminal and internal amino acid sequences of the purified enzyme were also detected in the deduced amino acid sequence (Fig. 1). The amino-terminal segment actually appeared to function as a signal sequence that was cleaved by a signal peptidase after the alanine residue at position 18. The calculated molecular mass of 25,004 Da (calculated from 693 bp coding for 231 amino acids, excluding the signal peptide that was removed by posttranslational modification) is in good agreement with the value of 25,000 estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein. From these results, we concluded that the ORF encoded the phosphotransferase gene. Computer sequence alignment using the SWISS-PROT and NBRF-PIR databases revealed that the gene appeared to be identical to the M. morganii PhoC acid phosphatase gene (accession no. P28581) (18). Seven bases in the ORFs were different, but these differences did not lead to differences in the predicted amino acid sequences: 54 A (in this report) to G (in phoC), 72 A to G, 276 A to T, 378 C to T, 420 T to G, 525 G to C, and 531 A to G.
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Reaction conditions for the synthesis of 5'-IMP by E. coli overproducing wild-type PhoC acid phosphatase.
Phosphotransferase activity was hardly detectable in E. coli
JM109(pUC18). On the other hand, the specific activity of
phosphotransferase in a cell extract of E. coli
JM109(pMPI501) cells grown in LB broth with induction by IPTG
was 1.25 ± 0.01 U/mg of protein; this value was 1.5- and 120-fold
higher than those of cells grown without IPTG induction [(8.50 ± 0.10) × 10
1 U/mg] and of M. morganii
[(1.04 ± 0.02) × 102 U/mg, without IPTG
induction], respectively. As the transformant showed high
phosphotransferase activity without IPTG induction, phoC
appeared to be expressed under the control of both its own promoter and
the lac promoter.
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), pH 4.0 (
), pH 5.0 (
), pH 5.5 (
),
and pH 6.0 (
). The reaction was carried out at 30°C in a reaction
mixture consisting of 0.1 M sodium acetate buffer containing (per
liter) 20 g of inosine (74.6 mM), 150 g of disodium hydrogen
pyrophosphate (200 mM), and 10 g (dry weight) of E. coli JM109(pMPI501) cells.
Random mutation of PhoC acid phosphatase and synthesis of 5'-IMP by E. coli overproducing mutated PhoC acid phosphatase. In order to suppress the dephosphorylation reaction and increase the efficiency of the transphosphorylation reaction, a random mutagenesis approach was used. By error-prone PCR, random mutations were introduced into the PhoC acid phosphatase gene. About 2,000 transformants that overexpressed mutated phoC were screened for increased yield of the phosphotransferase reaction. One candidate mutant, which showed almost the same phosphotransferase activity as E. coli(pMPI501) harboring wild-type phoC as well as lower 5'-nucleotidase activity, was successfully obtained in the first round. This improved variant, derived from E. coli JM109(pMPI600), was chosen as the parent for the second generation. The second round of random mutagenesis was performed on the mutated gene using the same procedure. About 3,000 transformants were screened, and a more improved mutant, E. coli JM109(pMPI700), was obtained. This candidate showed lower phosphotransferase activity but much lower 5'-nucleotidase activity than E. coli JM109(pMPI600).
The time course of 5'-IMP synthesis using E. coli overproducing the wild-type and mutated phoC gene products is shown in Fig. 3. Inosine was phosphorylated to 5'-IMP, along with the hydrolysis of PPi. Using E. coli JM109(pMPI501), 58.5 g of 5'-IMP per liter (111 mM) was synthesized, with a maximum molar yield of approximately 49%, from inosine. As the reaction time was prolonged, dephosphorylation was directed toward 5'-IMP and all of the synthesized 5'-IMP was hydrolyzed to inosine. With E. coli JM109(pMPI600), dephosphorylation of the synthesized 5'-IMP was suppressed to some extent, and a maximum of 82.9 g of 5'-IMP per liter (157 mM) was synthesized, with a molar yield of approximately 70%, from inosine.
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Characterization of mutated acid phosphatases. Gene sequencing of 1.2-kb EcoRI-HindIII fragments cloned into pMPI501, pMPI600, and pMPI700 showed that the only mutation site in phoC from pMPI600 was at Ile-171 (ATC), which was altered to Thr (ACC). In the gene from pMPI700, a further mutation was observed at Gly-92 (GGT), which was altered to Asp (GAT). In order to investigate the effect of a single G92D mutation, a G92D recombinant was constructed by site-directed mutagenesis.
The wild-type and I171T, I171T-G92D, and G92D mutant enzymes were purified from crude extracts of each E. coli JM109 transformant and analyzed. No changes in the levels of production of the wild-type and mutant enzymes in each E. coli transformant were observed under the culture conditions used. The kinetic constants of these enzymes in the transphosphorylation and dephosphorylation reactions are summarized in Table 1.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The M. morganii phosphotransferase gene was identical to the PhoC acid phosphatase gene (18). Although several phosphatase genes were also cloned from the M. morganii chromosomal DNA library in the course of the gene cloning, only the PhoC acid phosphatase exhibited phosphotransferase activity when PPi was used as the phosphate donor.
Acid phosphatases are further divided into three classes, designated A, B, and C, on the basis of amino acid sequence similarity. M. morganii PhoC acid phosphatase is classified as a class A nonspecific acid phosphatase; these enzymes have a polypeptide component of 25 to 27 kDa (19). To date, enzymes of this class have been isolated from several species, including Zymomonas mobilis, Salmonella typhimurium, and Shigella flexneri (14). Stukey and Carman have found that class A nonspecific acid phosphatases have a conserved sequence motif, KXXXXXXRP - (X12-54) - PSGH - (X31-54) - SRXXXXXHXXXD, which is shared by several lipid phosphatases and the mammalian glucose-6-phosphatases, and a mechanistic relationship among these enzymes has been suggested (17). Glucose-6-phosphatase is well known to be a multifunctional enzyme with potent phosphotransferase activity as well as phosphohydrolase activity (13). It catalyzes the six-position selective transfer of a phosphoryl group from PPi to glucose. It is interesting that the M. morganii PhoC acid phosphatase, which catalyzes the C-5'-position selective transfer of a phosphoryl group from PPi to nucleoside, shares a conserved sequence motif with glucose-6-phosphatase, as indicated in Fig. 1.
The reaction of phosphotransferase activity catalyzed by a phosphatase is thought to operate via a phosphate-enzyme intermediate. It involves the formation of binary enzyme-phosphoryl substrate complexes, which then dissociate to yield a common phosphoryl-enzyme intermediate. Transfer of the phosphoryl group to a phosphate acceptor leads to the production of a binary enzyme-phosphate acceptor-phosphate complex that ultimately dissociates to yield phosphorylated acceptor and free enzyme. Therefore, the phosphate acceptor competes with water, and a rather high concentration of acceptor is required for the transphosphorylation reaction.
As the Km value of the wild-type enzyme for inosine in the transphosphorylation reaction was extremely high, the efficiency of the transphosphorylation reaction was highly dependent on the concentration of inosine. The solubility of inosine in our reaction conditions was about 80 mM, reaching only two-thirds the Km value. Therefore, the efficiency of the transphosphorylation reaction of the wild-type enzyme was very low. On the other hand, the Km value for inosine of the I171T-G92D mutant enzyme was decreased to approximately one-third that of the wild-type enzyme, well below the achievable inosine concentration of 80 mM. As the affinity of the mutated enzyme for inosine increases, the efficiency of the transphosphorylation reaction should increase; additionally, the transphosphorylation reaction catalyzed by the mutated enzyme will proceed at a lower concentration of inosine than will that catalyzed by the wild-type enzyme. The mutations also led to a reduction in the Vmax of phosphotransferase activity, but the effect of the decrease in the Km was to allow a higher reaction rate under these conditions than would be expected from the reduced Vmax. In addition, as the Vmax of the 5'-nucleotidase activity of the mutated enzyme was decreased to approximately one-sixth the wild-type value, dephosphorylation of the synthesized 5'-IMP was suppressed. As the result of these changes in catalytic properties, the productivity of the mutated enzyme appeared to be much improved.
The I171T mutation was found to contribute to the decrease in the Km value for inosine and to the increase in the Km value for 5'-IMP. The G92D mutation did not seem to contribute to the decreased Km for inosine, but it contributed to the decreased Vmax values of both the phosphatase and the phosphotransferase activities. There was a synergistic effect of G92D combined with I171T: the Km value for inosine of the I171T-G92D mutant was reduced to about one-half that of the I171T single mutant.
We have demonstrated the potential of a new method of phosphorylating nucleosides using a mutated acid phosphatase. The 5'-nucleotidase activity of wild-type PhoC acid phosphatase rehydrolyzed the synthesized 5' nucleotide to a nucleoside, making the wild-type enzyme unsuitable for the phosphorylation of nucleosides. However, the catalytic properties of the enzyme could be much improved by a random mutagenesis approach, and the improved enzyme synthesized 5'-IMP at a practical level.
PPi is a safe and inexpensive compound that can be used in large excess. Also, PPi is easily synthesized from phosphate (16); therefore, an efficient phosphorylation process could be achieved by recycling PPi from phosphate formed as a by-product in the phosphorylation reaction. In addition, this enzyme shows broad substrate specificity for the phosphate acceptor (2) and could be applied to the synthesis of various 5' nucleotides. It was very interesting that the productivity of the enzyme in E. coli was much improved by the substitution of only two amino acid residues. Further studies on the structure-activity relationships of wild-type and mutated acid phosphatases are in progress.
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ACKNOWLEDGMENT |
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We thank T. Dairi, Toyama Prefectural University, for advice concerning this work.
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FOOTNOTES |
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* Corresponding author. Mailing address: Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan. Phone: 81-44-244-7138. Fax: 81-44-244-4757. E-mail: yasuhiro_mihara{at}ajinomoto.com.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, July 2000, p. 2811-2816, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
Kitagaki, H., Ito, K., Shimoi, H.
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