| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 97 === 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/�慊, 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�aC for 15 minutes, the residual activity of the enzyme was shown to be 30%.
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