Thermus thermophilus 是一種耐高溫的溫泉菌。耐高溫細菌體內的酵素除了有很好的熱穩定性，其對物理化學因子所造成的蛋白質變性也有很高的耐受性。Thermus thermophilus內的幾種基因，包括磷酸酶基因，被認為在生物科技應用價值上具有很大的潛力。
在本研究中將Thermus thermophilus的磷酸酶基因選殖至pET21b表現載體。然後將其轉殖至大腸桿菌中，以IPTG誘導其蛋白質表現，並利用Ni-NTA樹脂在變性或非變性條件下純化出磷酸酶蛋白質(30 kDa)。以變性條件下純化出的蛋白質做為抗原打入兔子體內以產生抗磷酸酶的抗體。另外，我們也用非變性純化出的酵素進行各種酵素活性的測試。
實驗結果顯示磷酸酶能在廣泛的溫度及pH值下作用，但其最適溫度是70C，而最適反應的pH值分別是：在酸性溶液中為pH 6、鹼性溶液中為pH 8。磷酸酶水解受質(p-nitrophenyl phosphate)時的動力學參數Km和Vmax分別為1.4 mM及0.67 mmol/min/g。此外，此一磷酸酶具有廣泛的磷酸酯受質分解能力。另外，此一磷酸酶對sodium tartrate具有耐受性；但其活性受到大部份的二價金屬離子所抑制。在高溫(80C, 15 min)處理後，其仍具30％的活性。

Thermus thermophilus is a thermophilic bacteria isolated from a thermal spring in Japan. This organism has considerable biotechnological potential as it can supply enzymes with thermal stability and better resistant to denaturing physical and chemical agents. Many new genes in Thermus thermophilus, including phosphatases, of potential interest for biotechnological applications were previously proposed in literature.
In this study, the gene encoding for a putative acid phosphatase from Thermus thermophilus HB8 has been cloned into pET21b expression vector and transformed into Escherichia coli BL21 (DE3) cells. The expression of the enzyme in the bacteria was induced by IPTG. The protein was purified from the cells using a Ni-nitrilo-tri-acetic acid agarose resin. Protein purified under denatured condition was used for production of polyclonal antibody in rabbit while protein purified under native condition was used for enzymatic activity assay to study the biochemical properties of this enzyme.
Our results showed that this enzyme is active within broad range of temperature and pH condition. The optimum temperature of acid phosphatase activity was found to be 70°C using p-nitrophenyl phosphate as substrate. The optimum pH of this enzyme was found to be at pH 6 in acidic buffer and pH 8 in alkaline buffer. The apparent Km and Vmax value of acid phosphatase for p-nitrophenyl phosphate was estimated to be 1.4 mM and 0.67 mmol/min/g, respectively. Besides that, this acid phosphatase enzyme was able to hydrolyse several phosphoesters compounds. This enzyme was inhibited by most metal ions but it was resistant to sodium tartrate. In terms of thermal stability, after heat treatment at 80C for 15 minutes, the residual activity of the enzyme was shown to be 30%.

Bilyeu K. D., Zeng, P. Coello, P., Zhang, Z. J., Krishnan, H. B. Bailey, A., Beuselinck, P. R. and Polacco J. C. 2008. Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial Phytase. Plant Physiol. 146: 468-477.

Boyce, A. and Walsh, G. 2007. Purification and characterization of an acid phosphatase with phytase activity from Mucor hiemalis Wehmer. J. Biotech. 132: 82-87.

Byeong C. J., Poole, P. S., Willis, A. C. and Macaskie, L. E. 1998. Purification and characterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp. Arch. Microbiol. 169: 166-173.

Dassa, E., Cahu, M., Desjoyaux-Cherel, B. And Boquet, P. L. 1982. The acid phosphatase with optimum pH of 2.5 of Escherichia coli. J. Biol. Biochem. 257: 6669-6676.

Du Plessis, E. M., Theron, J., Joubert, L., Lotter, T. and Watson, T. G. 2002. Characterization of a phosphatase secreted by Staphylococcus aureus strain 154, a new member of the bacterial class C family of nonspecific acid phosphatase. System. Appl. Microbiol. 25: 21-30.

Golovan, S., Wang, G., Zhang, J. and Forsberg, C. W. 2000. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can. J. Microbiol. 46: 59-71.

Guimaraes, L. H., Terenzi, H. F., Jorge, J. A., Leone, F. A., Polizeli, M. T. M. 2004. Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus. Biotechnol. Appl. Biochem. 49: 201-207.

Henne, A., Brüggemann, H., Raasch, C., Wiezer, A., Hartsch, T., Liesegang, H., Johann, A., Lienard, T., Gohl, O., Martinez-Arias, R., Jacobi, C., Starkuviene, V., Schlenczeck, S., Dencker, S., Huber, R., Klenk, H. P., Kramer, W., Merkl, R., Gottschalk, G. and Fritz, H. J. 2004. The genome sequence of the extreme thermophile Thermus thermophilus. Nature Biotechnol. 22: 547-553.

Huang, P-S. 2007. Translational enhancement of foreign protein expression by plant HSP101. Master Thesis. Appendix 1, page 123.

Kim, T. W. and Lei, X. G. 2005. An improved method for a rapid determination of phytase activity in animal feed. J. Anim. Sci. 83: 1062-1067.

Klabunde, T., Sträter, N., Fröhlich, R., Witzel, H. and Krebs, B. 1996. Mechanism of Fe(III)-Zn(II) purple acid phosphatase based on crystal structures. J. Mol. Biol. 259: 737-748.

Kurosawa, N., Fukuda, K., Itoh, Y. H. and Horiuchi, T. 2000. Partial purification and characterization of thermostable acid phosphatase from thermoacidophilic archaeon Sulfolobus acidocaldarius. Curr. Microbiol. 40: 57-60

Liu, B. L., Rariq, A., Tzeng, Y. M. and Rob, A., 1998. The induction and characterization of phytase and beyond. Enzyme Microb. Technol. 22: 415-424

Masui, R., Kurokawa, K., Nakagawa, N., Tokunaga, F., Koyama, Y., Shibata, T., Oshima, T., Yokoyama, S., Yasunaga, T. and Kuramitsu, S. 2004. Complete genome sequence of Thermus thermophilus HB8. Unpublished. NCBI database.

Nagai, Y. and Funahashi, S. 1962. Phytase (myo-inositol-hexaphosphate phosphohydrolase) from wheat bran. Part I. Purification and substrate specificity. Agric. Biol. Chem. 26: 794-803.

Niehaus, F., Bertoldo, E., Kähler, M. and Antranikian, G. 1999. Extremophiles as a source of novel enzymes for industrial application. Appl. Microbiol. Biotechnol. 51: 711-729.

Oddie, G. W., Schenk, G., Angel, N. Z., Walsh, N., Guddat, L. W., DeJersey, J., Cassady, A. I., Hamilton, S. E. and Hume, D. A. 2000. Structure, function and regulation of tartrate-resistant acid phosphatase. Bone 27: 575-584.

Oshima, T. and Imahori, K. 1974. Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa. Intl. J. Syst. Bacteriol. 24: 102-112.

Palacios, M. C., Haros, M., Rosell, C. M. and Sanz, Y. 2005. Characterization of an acid phosphatase from Lactobacillus pentosus: regulation and biochemical properties. J. Appl. Microbiol. 98: 229-237.

Pantazaki, A. A., Karagiorgas, A. A., Liakopoulou-Kyriakides, M. and Kyriakidis, D. A. 1999. Hyperalkaline and thermostable phosphatase in Thermus thermophilus. Appl. Biochem. Biotech. 75: 249-259.

Qian, H., Kornegay, E. T. and Denbow, D. M. 1996. Utilization of phytate phosphorus and calcium as influenced by microbial Phytase, cholecalciferol and the calcium: total phosphorus ratio in broiler diet. Poult. Sci. 76: 37-46.

Rossolini, G. M., Schippa, S., Riccio, M. L., Berlutti, F., Macaskie, K. E. and Thaller, M. C. 1998. Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology. Cell. Mol. Life Sci. 54: 833-850.

Saleh, M. T. and Belisle, J. T. 2000. Secretion of an acid phosphatase (SapM) by Mycobacterium tuberculosis that is similar to eukaryotic acid phosphatases. J. Bacteriol. 182: 6850-6853.

Schenk, G., Korsinczky, M. L. J., Hume, D., Hamilton, S. and DeJersey, J. 2000. Purple acid phosphatases from bacteria: similarities to mammalian and plant enzymes. Gene 255: 419-424.

Seung, H. K. and Kwang, K. C., et al. 2006. Cloning, sequencing and characterization of a novel phosphatase gene, phoI, from soil bacterium Enterobacter sp. 4. Curr. Microbiol. 52: 243-248.

Shimizu, M., 1993. Purification and characterization of phytase and acid phosphatase produced by Aspergillus oryzae K1. Biosci. Biotech. Biochem. 57: 1364-1365
Thaller, M. C., Schippa, S., Bonci, A., Cresti, S. and Rossolini, G. M. 1997. Identification of the gene (aphA) encoding the class B acid phosphatase / phosphotransferase of Escherichia coli MG1655 and characterization of its product. FEMS Microbiol Lett. 146: 191-198.

Tipton, K. F. 2002. Principles of enzyme assay and kinetic studies. In: Enzyme assays, 2nd ed., pp. 1-48. Eisenthal, R. and Danson, M. J. (eds), Oxford University Press, New York.

Ullar, A. H. J., Mullaney, E. M. and Dischinger, C. J. 1994. The complete primary structure elucidation of Aspergillus ficuum (niger), pH 6.0, optimum acid phosphatase by Edman degradation. Biochem. Biophys. Res. Commun. 203: 182-189.

Williams, R. A. D., Smith, K. E., Welch, S. G., Micallef, J. and Sharp, R. J. 1995. DNA relatedness of Thermus strains, description of Thermus brockianus sp. nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori). Intl. J. Syst. Bacteriol. 45: 495-499.

Yu, C. S., Lin, C. J. and Hwang J. K. 2004. Predicting subcellular localization of proteins for Gram-negative bacteria by support vector machine and peptide compositions. Protein Sci. 13: 1402-1406.

Zyla, K., Wikiera, A., Koreleski, J., Swiatkiewicz, S., Piironen, J. and Ledoux, DR. 2000. Comparison of the efficacies of a novel Aspergillus niger mycelium with separate and combined effectiveness of phytase, acid phosphatase, and pectinase in dephosphorylation of wheat-based feeds fed to growing broilers. Poultry Sci. 79: 1434-1443.