二胺基庚二酸去羧基酶之基因選殖與特性探討

自Propionibacterium acnes ATCC 6922菌中選殖出二胺基庚二酸去羧基酶(diaminopimelate decarboxylase，DAPDC)的基因，並在E. coli中建立蛋白表現系統。DAPDC蛋白的分子量為51,000 Da，屬於輔酶B6必需型酵素，對於反應受質有極高的專一性。本篇論文開發一種全新的DAPDC酵素活性分析方法，利用毛細管電泳分析技術來測量DAPDC之酵素動力學，此分析方法能更方便且快速地測量DAPDC酵素的活性。DAPDC酵素對反應受質及輔酶B6之Km值分別為1.6±0.3 mM與1.3±0.1 μM。另外，在測量DAPDC酵素與輔酶B6結合能力(Kd)上，本篇論文比較螢光光譜及微量透析兩種方法，螢光光譜法測得之Kd值為0.833 μM；微量透析法測得之Kd值為0.61±0.13 μM，兩種方法最後求得之Kd值也相當地接近。本論文的另一個主題，在探討Clostridium sticklandii菌株的離胺酸異構酶與輔酶B12 、B6的交互作用。實驗結果顯示，銨根離子會降低酵素與輔酶B12之Kd値，而輔酶B12結構中的ribonucleotide tail，為穩定結合輔酶B12所必須；另一方面，無論輔酶B6與反應受質是否存在，輔酶B12與其類似物對酵素之結合能力並未有明顯差異。

關鍵字

輔酶B6 ；二胺基庚二酸去羧基酶

並列摘要

A gene encoding diaminopimelate decarboxylase was cloned from Propionibacterium acnes ATCC 6922 and over-expressed in Escherichia coli. It encodes a protein of 476 amino acid residues with Mr 51,000. Diaminopimelate decarboxylase is a pyridoxal-5'-phosphate-dependent enzyme and is the only amino acid decarboxylase known to act on a carbon atom with D configuration in the substrate. A novel capillary electrophoresis-based enzyme assay method was established in this study. The Km for diaminopimelate and pyridoxal-5'-phosphate is 1.6±0.3 mM and 1.3±0.3 μM, respectively. The binding of pyridoxal-5'-phosphate to diaminopimelate decarboxylase was investigated by equilibrium dialysis and fluorescence spectroscopy. Similar results were obtained by both methods. The interactions between coenzymes and lysine aminomutase from Clostridium sticklandii was also investigated in this study. My results show that ammonia ion can stimulate the binding of coenzyme B12 to lysine aminomutase. The ribonucleotide tail of AdoCbl plays an important role in the binding of the cofactor. No significant difference in the binding of coenzyme B12 was observed, no matter whether coenzyme B6 or substrate analog is present or not.

並列關鍵字

diaminopimelate decarboxylase ； Propionibacterium acnes

參考文獻

[24]曾振興，離胺酸異構酶與鳥胺酸異構酶S次單元之交互作用，碩士論文，國立台北科技大學生物科技研究所，台北，2007。

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二胺基庚二酸去羧基酶之基因選殖與特性探討

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