| Summary: | 碩士 === 國立臺灣大學 === 生化科學研究所 === 93 === Deinococccus sp. NTU-1079 is isolated from Zhi-Sung hot spring in Taitung and found thermophilic as well as resistant to ionizing radiation. The gene of β-galactosidase from Deinococccus sp. NTU-1079 is cloned and the recombinant β-galactosidase is overexpressed in Escherichia coli, then simply purified by Ni-NTA column. The extracellular α-galactosidase is purified sequentially by DEAE sepharose, Superose 6 HR and HiTrap Q chromatography to homogeneity
Characterization experiments show that the two enzymes are stable under 50℃ for 3 h, and have the highest activities at neutral pH around 60 ~ 65℃. They are highly resistant to X-ray, under exposure to 2.58 KGy for 8 h, showing that the α-galactosidase and the β-galactosidase have remaining activity about 25% and 90%, respectively. Their Km and Vmax values for different substrates are also obtained. Furthermore, we find that the β-galactosidase belongs to glycoside hydrolase family 42 based on primary sequence alignment, and possesses similar structure to 4/7 superfamily enzymes according to the secondary and tertiary structure predictions. The tertiary structure of the β-galactosidase is built by computer modeling based on the known structure of β-galactosidase from Thermus sp. A4 as a template and the thermostability of the β-galactosidase may come from the formation of hydrogen bonds between subunits. The quaternary structure of the β-galactosidase is examined by gel filtration and sedimentation equilibrium and shows as dimer and trimer, respectively.
The transgalactosyl properties of these enzymes are applied to synthesize oligosaccharide derivatives as lectin-ligand probes. Using characteristics of thermostability, organic solvent-resistance, radiation resistance and specific catalytic mechanism of the enzymes, lectin-ligand probes can be established rapidly by the chemoenzymatic method.
|
|---|
