台灣嗜熱性奇異球菌NTU-1079之α-和β-半乳糖水解酶的研究及其於寡糖合成之應用

Abstract

Deinococccus sp. NTU-1079是由台東縣的栗松溫泉分離和鑑定的一株嗜熱性兼具抗輻射性的細菌。選殖它的β-半乳糖水解酶基因，並在大腸桿菌中讓它大量表現，再以鎳離子的親合性管柱純化之。同時，也依序利用DEAE sepharose、Superose 6 HR和HiTrap Q層析管柱，純化出它外泌性的α-半乳糖水解酶。 定性的結果顯示：此二酵素可穩定的存在於50℃的環境中；於60 ~ 65℃及中性pH值下有最好的活性，而且具有良好的輻射抗性。在以X射線，2.58 KGy劑量下照射達8小時，α-和β-半乳糖水解酶仍各別存留25%及90%的催化活性。在實驗中，我們也求得此二酵素對於不同受質的Km和Vmax值。更近一步，根據一級序列分析的結果，發現β-半乳糖水解酶屬於GH-42 family；二級和三級結構的預測和分析結果說明β-半乳糖水解酶屬於4/7 superfamily；以來自於Thermus sp. A4的β-半乳糖水解酶作為模版來做三級結構的電腦模擬，說明酵素的熱穩定性可能來自於次單元間氫鍵的作用力。利用膠體過濾層析和沉降平衡法來決定β-半乳糖水解酶的四級結構，分別得到二單元體及三單元體的結果。 在研究中，亦嘗試利用半乳糖水解酶的轉醣活性，將之應用於合成寡糖衍生物 ─ 凝集素的探針上。利用嗜熱性菌酵素之耐熱性、對有機溶劑的抗性、輻射抗性和酵素催化之專一性，省略傳統有機化學合成法過於繁複的醣化合成步驟，已達成快速建立凝集素的探針資料庫的目的。

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.