L-天冬胺酸4-去羧酶特性分析與調控雙功能酵素活性中重要胺基酸之研究

Abstract

本研究自革蘭氏陰性細菌Pseudomonas sp. ATCC 19121 選殖 L-天冬胺酸4-去羧酶(Asd)基因群，成功地於大腸桿菌宿主中表現帶有His-tagged 的可溶重組蛋白，並完成此重組蛋白之特性分析，及其雙功能角色中部份重要胺基酸的研究。Asd 為工業生產丙胺酸的重要酵素，選殖酵素基因長1,593 bp，可轉譯出59,243 Da 之酵素蛋白；Asd 蛋白質序列與其他Asd 之相同性為37-85%，與部份轉胺酶則為39-63%，asd基因在演化上與革蘭氏陽性菌者不同。次選殖asd 至pET 表現系統，建構之Escherichia coli BL21(DE3)pLysS/pES1，經0.4 mM IPTG 誘導5小時，胞內酵素以親和層析管柱純化，產率為33 mg/L，酵素比活性最高可達280 U/mg；動力學分析純化酵素Km 及 Vmax 分別為11.50 mM、0.11 mM/min。測試的二價金屬離子皆抑制酵素活性。此酵素反應活性最佳的條件為45℃、pH 5.0，且酵素在pH 5~7 的穩定性佳；膠體過濾分析實驗中，當移動相之緩衝液由pH 5.0 提高至pH 7.0 時，4.4%的重組酵素由12 聚合體分解為雙體；在pH 8.0 的tris 緩衝液中培養，原子力顯微鏡觀察Asd以雙體形式存在。此重組酵素對D,L-Asp, L-Glu, L-Gln 及 L-Ala 有微量轉胺活性，以L-Asp 為基質時，酵素主要的去羧活性為轉胺活性的2,477 倍。依據與轉胺酶之多序列比對，建構的16 個Asd 突變株，成功利用pET 表現系統大量表現重組蛋白，除AsdT319Y 及AsdD350S 外皆為胞內可溶蛋白，且純化酵素皆可為Asd 原態酵素抗體辨識。其中AsdF203W 突變蛋白，轉胺活性較原態酵素提高37.74%，去羧酶活性減少27.1%；AsdM177L 兩種活性皆提升約16~25%；AsdH336R、AsdD359P 及AsdV474L 去羧活性提高1.7~2.6 倍，轉胺活性下降，對於β-去羧反應的專一性較原態酵素提升。

The L-aspartate 4-decarboxylase (Asd) gene was cloned from Pseudomonas sp. ATCC 19121, a gram negative bacterium, and expressed as a His-tagged fusion protein in Escherichia coli BL21(DE3)pLysS. Characterization of the recombinant protein was performed in this study. The 1,593-bp asd encodes a protein with a molecular mass of 59,243 Da. Its protein sequence shares 37-85% identity upon other Asds and 39-63% upon some aminotransferases. The asd diverged in evolution from those in gram positive strains. Productivity rate of the C-terminal His-tagged Asd was at 33 mg/L of E. coli transformant culture. The kinetic parameters Km and Vmax of the fusion protein for L-aspartate were 11.50 mM and 0.11 mM/min, respectively. All divalent ions inhibited Asd activity in our study. Optimum temperature for Asd reaction was 45℃. The recombinant Asd exhibited its maximum activity in β-decarboxylation at pH 5.0 and specific activity of 280 U/mg. This enzyme is stable in the pH range of 5 to 7, and 4.4% protein dissociated into dimer from dodecamer when the pH shifted from 5.0 to 7.0 in gel filtration analysis. The recombinant Asd displayed little aminotransferase activity when D,L-Asp, L-Glu, L-Gln and L-Ala were served as substrates. Asd catalyzed the β-decarboxylation of L-aspartate 2,477 times more than transamination reaction. According to the multiple alignment of protein sequences of Asd and aminotransferases, 16 site-directed mutants of Asd were designed and constructed. Proteins were purified from cell lysates except AsdT319Y and AsdD350S, which form inclusion bodies, and analyzed by SDS-PAGE. All the mutant proteins were recognized by polyclonal antibodies of Asd. AsdF203W mutant protein exhibited aminotransferase activity 37.74% higher than the native one, and decreased 27.1% of the decarboxyalse activity. AsdM177L enhanced 16-25% for each activity. AsdH336R, AsdD359P and AsdV474L enhanced decarboxylase activity 1.7 to 2.6 times higher than the native protein; meanwhile, they showed decreased aminotransferase activity. As a result, these 3 mutant proteins are more specific in β-elimination reaction than the native Asd.