嗜熱菌Pyrococcus abyssi GE5之新穎高耐熱麩胺酸脫羧酶之基因選殖與重組酵素的催化特性

麩胺酸脫羧酶催化L-麩胺酸,去羧以產生γ-胺基丁酸及二氧化碳。麩胺酸脫羧酶可以在許多生物中被發現，如單細胞生物、原核生物和古生菌，上至植物和動物。γ-胺基丁酸被普遍認為在哺乳動物的中樞神經系統起主要抑制性神經元的作用，並和糖尿病、精神分裂症、躁鬱症和帕金森病密切關聯。此外，γ-胺基丁酸是吡咯烷酮的前體，其單體被工業利用以合成尼龍4聚合物，因此，麩胺酸脫羧酶適合用於生物技術和商業來生產γ-胺基丁酸。Pyrococcus abyssi GE5 （P. abyssi）是超嗜熱古生菌，並棲息位於火山周圍的海底熱噴泉。P. abyssi的最佳生存溫度大約介於96 ℃左右。一個擁有1173編碼，假定為麩胺酸脫羧酶的cDNA片段成功從P. abyssi複製出來，並表示為rPaGAD。我們成功於大腸桿菌（BL -21 DE3）表達系統表達該基因及純化到擁有活性的rPaGAD。催化檢測表明麩胺酸脫羧酶是一種在100℃下仍舊保有活性的熱穩定性酶。rPaGAD最適pH值為pH 5 。其催化活性通過γ-胺基丁酸的形成作為最終產物進行檢測，並使用高效液相色譜法（HPLC ）內設的紫外線 /可見光檢測器定量確認。Cluster Omega，一個多序列比對工具顯示麩胺酸脫羧酶存在一些較為穩定不變的胺基酸，其不變性可以在植物、革蘭氏陰性和嗜熱單細胞生物體的麩胺酸脫羧酶中發現。特別是離胺酸237（Lys）, 它是高度保留的，並與磷酸吡哆醛（PLP）连结，從而活化麩胺酸脫羧酶。生物演化圖譜聚集起源密切相似的麩胺酸脫羧酶，並疏遠屬於不同物種的麩胺酸脫羧酶。

關鍵字

麩胺酸脫羧酶； γ-胺基丁酸；高效液相色譜法；磷酸吡哆醛

並列摘要

Glutamate decarboxylase (GAD) catalyzes the decarboxylation of L-glutamate/glutamic acid to produce gamma-aminobutyric acid (GABA) and carbon dioxide (CO2). GAD can be found in many biological life forms, ranging from unicellular organisms such as prokaryotes and archaeon to complex plants and animals. GABA is known as major inhibitory neurotransmitter in mammalian central nervous system (CNS) and closely associated with diabetes, schizophrenia, bipolar disorder and Parkinson disease. Apart from that, GABA is the precursor of 2-pyrrolidone, a monomer for industrial synthesized polymeric nylon 4. Thus, GAD is useful for the production of GABA for biotechnological and commercial applications. Pyrococcus abyssi GE5 (P. abyssi) is a hyperthermophilic archaeon inhabits at hydrothermal vents that are commonly located around volcanically areas. P. abyssi has optimal surviving temperature around 96 degree Celcius (°C). A cDNA fragment of 1173bp encoding a putative GAD enzyme was cloned from P. abyssi, denoted as rPaGAD. We successfully expressed the gene in Escherichia coli BL-21 (DE3) competent cell expression system and purified the active recombinant rPaGAD enzyme. Catalytic properties showed it is a highly thermostable enzyme which remained active at 100°C. The optimal pH of the rPaGAD is pH 5. Its catalytic activity was confirmed by the formation of GABA as end product that analyzed and quantitated using high performance liquid chromatography (HPLC) with UV/Vis detectors. Cluster Omega, a multiple sequence alignment tool revealed several conserved amino acids exist within GADs of plant, gram-negative and thermophilic unicellular organisms, especially lysine237 (Lys). It is highly conserved, forming Schiff-base with pyridoxal phosphate (PLP), an active form of vitamin B6 that activates apoenzyme into holoenzyme. Phylogenetic tree clustered GADs of similar origins closely to each other while distancing GADs of distinct species.

並列關鍵字

glutamate decarboxylase ； gamma-aminobutyric acid ； Pyrococcus abyssi ； high performance liquid chromatography ； pyridoxal phosphate

參考文獻

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[7]J. Kim, J.P. Kershner, Y. Novikov, R. K. Shoemaker, and S. D. Copley, "Three serendipitous pathways in E. coli can bypass a block in pyridoxal-5'-phosphate synthesis," Mol Syst Biol. 1038, 2010.

[9]Available: http://www.uniprot.org/uniprot/Q9LSH2

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嗜熱菌Pyrococcus abyssi GE5之新穎高耐熱麩胺酸脫羧酶之基因選殖與重組酵素的催化特性

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