以His-tag固定D-型胺基酸氧化酶之性質探討

Properties of D-amino acid oxidase immobilized through His-tag

廖仁傑(Liao-Ren Jie)

指導教授 ： 游吉陽

大同大學/工程學院/生物工程學系所/碩士(2007年)

摘要

D-型胺基酸氧化酶(D-amino acid oxidase；EC 1.4.3.3；DAO)為一種以FAD為輔基之氧化還原酵素，能專一性催化D-型胺基酸進行氧化脫氨作用，產生相對之α-keto acid，並有ammonia及過氧化氫伴隨著產生。工業上DAO常用於生產7-ACA。DAO之固定化研究常以化學法為主，因此，本論文表現及純化於C-terminus帶有His-tag之Rhodosporidium toruloides DAO (RtDAO)及Trigonopsis variabilis DAO (TvDAO)，利用His-tag與Ni2+產生之配位鍵，分別將RtDAO及TvDAO固定於Ni-NTA Magnetic Agarose Beads之上。探討溫度、pH值、H2O2對於液相及固定相酵素活性之影響，及分別針對動力參數、儲存穩定性、冷凍-解凍循環、固定化酵素之重複使用性進行分析。結果顯示RtDAO固定後其酵素熱穩定性、H2O2耐受性及於室溫下之儲存穩定性，皆有明顯之改善。於50℃下加熱1小時後，液相RtDAO完全失去活性，固定化RtDAO活性仍保有56%。H2O2耐受性方面，與10 mM H2O2反應9小時後，液相RtDAO殘餘活性為21.8%，而固定化RtDAO殘餘活性則為72%；即使經過15小時後，固定化RtDAO活性則仍有35.2%。儲存穩定性方面，於室溫下儲存10天後，固定化RtDAO其殘餘活性為73.5%，而液相RtDAO則失去活性。至於TvDAO固定後與液相酵素相比，其溫度、pH值、H2O2耐受性、儲存穩定性及冷凍-解凍循環則皆未有明顯之改善。

關鍵字

D-型胺基酸氧化酶 ； His-tag

並列摘要

D-amino acid oxidase (DAO; EC 1.4.3.3) is one of the dehydrogenase-oxidase enzymes containing FAD as the prosthetic group. DAO catalyzes the specific oxidation of D-amino acids to produce the corresponding α-keto acids with hydrogen peroxide and ammonia as by-products. The most important industrial application of DAO is the production of 7-aminocephalosporanic acid (7-ACA). The immobilization of DAO is often performed using chemical methods. In this thesis, recombinant Rhodosporidium toruloides DAO (RtDAO) and Trigonopsis variabilis DAO (TvDAO) fused C-terminus His-tags were expressed and purified. RtDAO and TvDAO were then immobilized on Ni-NTA Magnetic Agarose Beads through the interaction between His-tag and Ni2+. The effects of temperature, pH, and the presence of hydrogen peroxide were studied for both free and immobilized enzymes. In addition, enzyme kinetics, storage stability, reusability of the immobilized DAO were also discussed. The immobilized RtDAO exhibited significant improvements in stability against thermal and hydrogen peroxide inactivation. After incubation at 50℃ for 1 h, the relative activity of the immobilized RtDAO was 56% while the free RtDAO was completely inactivated. In the presence of 10 mM hydrogen peroxide, the residual activities of free and immobilized RtDAO after 9 h incubation were 21.8 and 72%, respectively. The immobilized RtDAO still retained 35.2% activity even after 15 h of incubation. After storage at room temperature for 10 d, the residual activity of immobilized RtDAO was 73.5% compared to the total loss of activity of the free enzyme. No improvements in stability were observed for immobilized TvDAO.

並列關鍵字

His-tag ； DAO

參考文獻

高緒鈞，以固定化技術改善D-型胺基酸氧化酶之穩定度，2007，碩士論文，生物工程研究所，大同大學，臺北。

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