綠竹蘋果酸去氫酶之表現與檢定

Abstract

蘋果酸去氫酶 (malate dehydrogenase, MDH EC 1.1.1.37) 在生物體內分佈非常廣泛，負責可逆性催化oxaloacetate 和malate間的轉換。高等植物中依照對不同輔酶的專一性、胞內位置和生理功能，而有不同的異構酶，其中利用NAD和位於乙醛酸循環體 (glyoxysome) 的gMDH是一種植物中尚未被完全研究的MDH。 本實驗室之前於綠竹cDNA庫篩選細胞分裂素氧化酶/去氫酶時，意外篩到了一可能是綠竹gMDH的基因，因而在此篇論文裡我們將對此基因進行選殖、原核表現和檢定。回推基因庫發現此gMDH不具任何內插子，而蛋白質序列分析則顯示和許多其他植物的gMDH有高相似性，其中和同為禾本科之稻米相似度更達96%。在N端接上Trx-His tag並透過大腸桿菌BL21 (DE3) 表現出約57 kD的重組蛋白質，也顯示了高度MDH活性，在在都確認了此一基因即綠竹乙醛酸循環體的MDH。 以重組蛋白質製備MDH多株抗體後，從綠竹筍中直接純化MDH。經緩衝液粗抽、硫酸銨分劃、親和層析法 (Blue Sepharose¬¬¬ CL-6B)、膠體過濾法 (Superose 12 HR 10/30) 得到的純化蛋白質，和重組蛋白質在動力學和最適反應pH上有相似的性質：對malate kcat/Km約150、對NAD+ 約2500，最適反應pH為9.5。但在最適反應溫度上，重組MDH偏好低溫環境而純化MDH卻偏好高溫。以膠體過濾法測得MDH原態分子量介於130~115 kD，次單元分子量由SDS-PAGE得約45 kD，推測應為同質二元體結構。

Malate dehydrogenase (MDH EC 1.1.1.37), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Glyoxysomal NAD-dependent MDH (gMDH) is one class of MDH that has not been extensively characterized in plants, and also there’s no any research studying the relation between gMDH and cytokinin oxidase/dehydrogenase (CKX, EC 1.55.99.12). Unexpectedly, a putative Mdh cDNA was screened with the specific probe of CKX from the cDNA library of Bambusa oldhamii in our laboratory. Here we present the cloning, characterization and prokaryotic expression of this putative Mdh. Sequence alignment shows that there’s a high homology between the deduced amino sequence of BogMDH and MDH protein in glyoxysome in Oryza sativa (96%). Nearly 57 kD fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the protein. All these results suggest that BogMDH encodes a glyoxysomal MDH. Screened the genomic library indicated the Mdh has no intron. After preparing the polyclonal antibody by using fusion protein as the antigen, we partial purified MDH from bamboo through buffer extraction, ammonium sulfate precipitation, and Fast Protein Liquid Chromatography (FPLC). The recombinant MDH and the purified MDH both have similar characteristics in kinetics and the optimum pH. But the recombinant protein prefers a low temperature. In contrast, the purified protein favors a higher temperature. Using Superose 12 column, the molecular weight of native form bamboo MDH was estimated to be 130 ~ 115 kD, and the subunit form was about 45 kD by SDS-PAGE. It might be a homodimeric enzyme.