探討嗜熱菌 Meiothermus taiwanensis ATCC BAA-400 之半乳糖激酶酵素動力學及應用於合成 Pk 抗原類似物

Kinetics Studies with Galactokinase from Meiothermus taiwanensis ATCC BAA-400 and Application of Galactokinase to Synthesize Pk-antigen analoge

李泗芃

指導教授 ： 林俊成

國立清華大學/理學院/化學系/碩士(2012年)

摘要

在本論文中，選殖、表達台灣本土嗜熱菌 Meiothermus taiwanensis ATCC BAA-400 的半乳糖激酶 （galactokinase，GalK） 並以 IMPACTTM-CN系統純化可得 16 mg/L 的半乳糖激酶重組蛋白。半乳糖激酶重組蛋白最佳化反應溫度為 75 oC、最佳化反應酸鹼值為 9.0。半乳糖激酶重組蛋白對半乳糖的kcat/Km 值為 168.47 s-1mM-1，於 55 oC 和 75 oC 分別測得的比活性為 240 U/mg 和 388 U/mg。半乳糖激酶與實驗室已有的葡萄糖-1-磷酸胸苷轉移酶 （glucose-1-phosphate thymidylyltransferase，RmlA）結合，可進行一鍋化反應製備尿苷二磷酸半乳糖 （UDP-Gal）。
分別由 Neisseria meningitides 與 Haemophilus influenzae Rd KW20 選殖兩種來源不同的 α-1,4-半乳糖轉移酶。C-端截斷 （truncated） 19 個胺基酸的N. meningitides α-1,4-半乳糖轉移酶（LgtC-19）與N-端截斷 38 個胺基酸的 H. influenzae Rd KW20 α-1,4-半乳糖轉移酶（N-cut-38-Hi0258），皆可運用於階段式一鍋化反應合成 Pk 抗原類似物 （α-D-Gal-(1-4)-β-D-Gal-(1-4)-β-D-Glc -OC6H12N3）。

關鍵字

半乳糖激酶 ； 半乳糖轉移酶 ； Pk 抗原

並列摘要

In this study, the recombinant galactokinases (GalK) from Meiothermus taiwanensis ATCC BAA-400 was cloned, over-expressed, and purified by IMPACTTM-CN system with a yield of 16 mg/L cell cultures. The optimal reaction conditions for recombinant GalK are at 75 °C, and pH at 9.0. The kcat/Km value of recombinant GalK toward galactose is 168.47 s−1 mM−1 and specific unit at 55 °C and 75 °C are 240 U/mg and 388 U/mg, respectively. The GalK was combined with glucose-1- phosphate thymidylyltransferase (RmlA) to synthesize uridine 5’-diphosphate galactose (UDP-Gal) in one-pot reaction.
In addition, the genes of α-1,4-galactosyltransferases (GalTs) from Neisseria meningitides (19 residues at C-terminal deleted; LgtC-19) and Haemophilus influenzae Rd KW2 (38 residues of N-terminal was deleted;N-cut-38-Hi0258) were cloned and the corresponding proteins were over-expressed. Both α-1,4-GalTs were respectively combined with GalK and RmlA to synthesize Pk antigen derivatives (α-D-Gal-(1-4)-β-D -Gal-(1-4)-β-D-Glc-OC6H12N3) in sequential addition one-pot reaction manner.

並列關鍵字

Galactokinase ； Galactosyltransferase ； Pk antigen

參考文獻

1. Koeller, K. M.; Wong, C. H., Enzymes for chemical synthesis. Nature 2001, 409, 232-240.

3. Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B., Industrial biocatalysis today and tomorrow. Nature 2001, 409, 258-268.

4. Schoemaker, H. E.; Mink, D.; Wubbolts, M. G., Dispelling the myths--biocatalysis in industrial synthesis. Science 2003, 299, 1694-1697.

5. Varki, A., Nothing in glycobiology makes sense, except in the light of evolution. Cell 2006, 126, 841-845.

6. Ohtsubo, K.; Marth, J. D., Glycosylation in cellular mechanisms of health and disease. Cell 2006, 126, 855-867.