Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237
Description of the subject. Alkaline phosphatases (APases) are commonly used as nonradioactive markers for detecting specific proteins or DNA targets in clinical medicine and molecular biology. However, their applications in the biotechnology industry require thermostability and storage stability. O...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Presses Agronomiques de Gembloux
2017-01-01
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| Series: | Biotechnologie, Agronomie, Société et Environnement |
| Subjects: | |
| Online Access: | http://hdl.handle.net/11006/272 |
| _version_ | 1818939255770054656 |
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| author | Nguyen, HC. Wu, SP. Su, CH. Hwang, TS. |
| author_facet | Nguyen, HC. Wu, SP. Su, CH. Hwang, TS. |
| author_sort | Nguyen, HC. |
| collection | DOAJ |
| description | Description of the subject. Alkaline phosphatases (APases) are commonly used as nonradioactive markers for detecting specific proteins or DNA targets in clinical medicine and molecular biology. However, their applications in the biotechnology industry require thermostability and storage stability. Our preliminary study revealed that APase from Thermus sp. NTU-237 (TsAPase) is thermostable and exhibits high activity. Therefore, it is desirable to establish the optimal conditions for its application. Objectives. To characterize APase from thermophile Thermus sp. NTU-237 and to evaluate its potential applications. Method. The APase gene of Thermus sp. NTU-237 was cloned and expressed in Escherichia coli. Subsequently, the effects of buffer, glycerol, sodium dodecyl sulfate (SDS), NaCl, and temperature on enzyme activity were studied to establish the optimal conditions for TsAPase assays. In addition, the potential for application of TsAPase was evaluated using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) active staining method. Results. Recombinant TsAPase was identified as having a dimeric structure and a molecular mass of 109 kDa. Sequence alignment analysis of known thermophilic APases with TsAPase and E. coli APase revealed that catalytic and binding residues were highly conserved. This finding suggested that the catalytic mechanism of TsAPase is the same as that of other APases. TsAPase activity was inhibited by glycerol and SDS but enhanced by NaCl and Tris-HCl buffer. The kinetic parameters Km, kcat, and Vmax were determined to be 81 µM, 6.08 s-1, and 6.76 U·mg-1, respectively. The optimum temperature of TsAPase was 80 °C, and TsAPase was stable at room temperature for more than 10 days. Moreover, TsAPase could catalyze the dephosphorylation of BCIP and could elicit blue color development by the BCIP/NBT reaction kit at room temperature. Conclusions. These results illustrate that TsAPase has potential for application in medical or basic research. |
| first_indexed | 2024-12-20T06:20:51Z |
| format | Article |
| id | doaj.art-dc33b609a3ee464496eb3636925ae905 |
| institution | Directory Open Access Journal |
| issn | 1370-6233 1780-4507 |
| language | English |
| last_indexed | 2024-12-20T06:20:51Z |
| publishDate | 2017-01-01 |
| publisher | Presses Agronomiques de Gembloux |
| record_format | Article |
| series | Biotechnologie, Agronomie, Société et Environnement |
| spelling | doaj.art-dc33b609a3ee464496eb3636925ae9052022-12-21T19:50:25ZengPresses Agronomiques de GemblouxBiotechnologie, Agronomie, Société et Environnement1370-62331780-45072017-01-01212117126Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237Nguyen, HC.Wu, SP.Su, CH.Hwang, TS.Description of the subject. Alkaline phosphatases (APases) are commonly used as nonradioactive markers for detecting specific proteins or DNA targets in clinical medicine and molecular biology. However, their applications in the biotechnology industry require thermostability and storage stability. Our preliminary study revealed that APase from Thermus sp. NTU-237 (TsAPase) is thermostable and exhibits high activity. Therefore, it is desirable to establish the optimal conditions for its application. Objectives. To characterize APase from thermophile Thermus sp. NTU-237 and to evaluate its potential applications. Method. The APase gene of Thermus sp. NTU-237 was cloned and expressed in Escherichia coli. Subsequently, the effects of buffer, glycerol, sodium dodecyl sulfate (SDS), NaCl, and temperature on enzyme activity were studied to establish the optimal conditions for TsAPase assays. In addition, the potential for application of TsAPase was evaluated using the 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) active staining method. Results. Recombinant TsAPase was identified as having a dimeric structure and a molecular mass of 109 kDa. Sequence alignment analysis of known thermophilic APases with TsAPase and E. coli APase revealed that catalytic and binding residues were highly conserved. This finding suggested that the catalytic mechanism of TsAPase is the same as that of other APases. TsAPase activity was inhibited by glycerol and SDS but enhanced by NaCl and Tris-HCl buffer. The kinetic parameters Km, kcat, and Vmax were determined to be 81 µM, 6.08 s-1, and 6.76 U·mg-1, respectively. The optimum temperature of TsAPase was 80 °C, and TsAPase was stable at room temperature for more than 10 days. Moreover, TsAPase could catalyze the dephosphorylation of BCIP and could elicit blue color development by the BCIP/NBT reaction kit at room temperature. Conclusions. These results illustrate that TsAPase has potential for application in medical or basic research.http://hdl.handle.net/11006/272Thermusthermostablealkaline phosphatasecharacterizationchromogenicapplicationenzymology |
| spellingShingle | Nguyen, HC. Wu, SP. Su, CH. Hwang, TS. Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 Biotechnologie, Agronomie, Société et Environnement Thermus thermostable alkaline phosphatase characterization chromogenic application enzymology |
| title | Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 |
| title_full | Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 |
| title_fullStr | Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 |
| title_full_unstemmed | Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 |
| title_short | Biochemical, biophysical, and thermal properties of alkaline phosphatase from thermophile Thermus sp. NTU-237 |
| title_sort | biochemical biophysical and thermal properties of alkaline phosphatase from thermophile thermus sp ntu 237 |
| topic | Thermus thermostable alkaline phosphatase characterization chromogenic application enzymology |
| url | http://hdl.handle.net/11006/272 |
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