Rhodosporidium toruloides與Trigonopsis variabilis D型胺基酸氧化酶以基因融合與固定化增加穩定性

STABILIZATION OF D-AMINO ACID OXIDASES FROM RHODOSPORIDIUM TORULOIDES AND TRIGONOPSIS VARIABILIS BY GENE FUSION AND IMMOBILIZATION

王詩瑞(Shih-Juei Wang)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/博士(2008年)

摘要

酵母菌 Rhodosporidium toruloides與 Trigonopsis variabilis 之D型胺基酸氧化酶 (D-Amino acid oxidases; DAO) 是一種以FAD為輔基的homodimeric flavoenzymes。 利用六個核苷酸分別連接這兩種DAO的cDNA 基因並轉殖於E. coli 中作異源表現，產生僅含單一多胜肽鏈之對應double DAOs，即dRtDAO 與 dTvDAO。dRtDAO、dTvDAO在比活性上與RtDAO及TvDAO相似，且在最適pH與pH穩定性上皆維持於pH值8.5。在熱穩定性分析方面，dRtDAO的Tm 值較RtDAO 的上升5℃，而 dTvDAO 的Tm 值只比TvDAO 的提升2℃。利用biotin與streptavidin 的鍵結特性，將酵素固定於streptavidin-coated beads後，發現在熱穩定性上可更進一步獲得改善。比較固定化與未固定化double DAO的Tm 值，發現前者的Tm 值上升約6℃。在過氧化氫耐受性上，酵素以10 mM H2O2 處理後，dRtDAO、dTvDAO的半生期分別為60與45 分鐘，較RtDAO、TvDAO的半生期分別增加約2及1.5倍。經固定化後，double DAOs在氧化耐受性方面亦有提昇。同樣以10 mM H2O2 處理後，固定化dRtDAO、dTvDAO的半生期分別為10 與5 小時，與未固化的半生期相比，增加約10及7倍。

關鍵字

D型胺基酸氧化酶 ； 基因融合 ； 固定化 ； 酵母菌Rhodosporidium toruloides ； 穩定性 ； 酵母菌Trigonopsis variabilis

並列摘要

D-Amino acid oxidases from yeast Rhodosporidium toruloides and Trigonopsis variabilis (RtDAO and TvDAO) are both homodimeric flavoenzymes. Two of their cDNA genes were connected by a hexanucleotide linker and heterologously expressed in E. coli to produce the corresponding double DAOs (dRtDAO and dTvDAO) made of only a single polypeptide. The specific activities of double DAOs remained similar to those of native dimeric DAOs. The optimal pH for activity and the best pH stability of double DAOs were both observed at pH 8.5. The Tm value for dRtDAO was shifted 5℃ higher while that for dTvDAO was increased only by 2℃, in comparison with the corresponding native counterparts. The improvement in thermal stability was further obtained by immobilization through the biotin-streptavidin binding onto streptavidin-coated beads. Immobilized double DAOs had 6℃ higher Tm values than their soluble counterparts. In the presence of 10 mM H2O2, dRtDAO and dTvDAO exhibited half-lives of about 60 and 45 min, respectively, which were 2 and 1.5 folds more stable than their native DAOs, respectively. The oxidative stability of double DAOs was also further enhanced after immobilization. The immobilized dRtDAO and dTvDAO exhibited half-lives of about 10 and 5 h, respectively, which gave 10- and 7-fold, greater stability than their soluble forms under the same oxidative condition.

並列關鍵字

D-amino acid oxidase ； gene fusion ； immobilization ； Rhodosporidium toruloides ； stabilization ； Trigonopsis variabilis

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