以SILAFFIN引導仿生矽化固定TRIGONOPSIS VARIABILIS D型胺基酸氧化酶

IMMOBILIZATION OF TRIGONOPSIS VARIABILIS D-AMINO ACID OXIDASE VIA SILAFFIN-MEDIATED BIOMIMETIC SILICIFICATION

陳怡靜(Yi-Jing Chen)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2012年)

摘要

以仿生矽化固定酵素已被廣泛的研究探討，本研究分別使Trigonopsis variabilis DAO (TvDAO)的N和C端融合His tag和二氧化矽沉澱胜肽R4或R5，產生融合蛋白HTvDAOR4及 HTvDAOR5，以自行進行仿生矽化作用。SDS-PAGE分析清楚顯示，大腸桿菌表現之重組TvDAO融合蛋白分子量如預期約42 kDa。經金屬螯合層析純化後，檢測得比活性分別約51、49 U/mg。經過包覆條件的微調後，最適的仿生矽化條件乃在100 l反應體積中，將50 mM磷酸鹽緩衝液 (pH 8)、400 g磁性奈米粒子、10 g融合TvDAO和0.1 M 經水解的tetramethoxysilane (TMOS) 依次混合後而得。相較於未融合固定的原始HTvDAO，固定HTvDAOR4和HTvDAOR5最適反應pH由7.5變為8.0。前者於pH＞5而後者於pH＞8時有較佳穩定性。最適反應溫度皆由40oC變為55oC，其TM值則分別為59和60oC，亦優於HTvDAO的53oC。至於H2O2氧化抵抗能力，經固定的融合TvDAO之半衰期約61~67分鐘，為HTvDAO之1.4~1.6倍。固定融合TvDAOR4/R5 20次重複操作後分別殘留89和66%之活性，因此以融合R4或R5胜肽的TvDAO形式進行仿生矽化包埋不僅可增加其pH和熱穩定性，同時提升對H2O2的氧化耐受性。

關鍵字

D型胺基酸氧化酶 ； 仿生矽化

並列摘要

Enzyme immobilization by biomimetic silicification have been extensively investigated. In this study, Trigonopsis variabilis D-amino acid oxidase (TvDAO) was N and C-teriminally fused to His tag and silica-precipitating peptide R4 or R5, respectively, to generate fusion protein HTvDAOR4 and HTvDAOR5 for auto-silicification. SDS-PAGE analysis clearly showed that the molecular weights of the recombinant TvDAO fusion proteins expressed in E. coli were both approximately 42 kDa as expected. After purification by metal chelation chromatography, the specific activities of HTvDAOR4 and HTvDAOR5 were determined to be 51 and 49 U/mg, respectively. After fine adjustment of the conditions for capsulation, the optimal biomimetic silification was achived by mixing 50 mM potassium phosphate buffer (pH 8), 400 g magnetic nanoparticle, 10 g fused TvDAO and 0.1 M hydrolyzed tetramethoxysilane in order in a 100 l reaction volume. As compared to free unfused HTvDAO, the optimal pH of immobilized HTvDAOR4 and HTvDAOR5 were shifted from 7.5 to 8.0. The former exhibited better pH stability at pH＞5, while the latter at pH＞8. The optimal temperature of immobilized and fused HTvDAO were both shifted from 40oC to 55oC. They had the TM values of 59 and 60oC, respectively, superior to 53oC for TvDAO. With regard to the oxidative resistance to H2O2, both immobilized TvDAO fusion proteins showed half-live increased by 1.4 to 1.6 folds which were of 61 and 67 min. After 20 cycles of repeated operation, immobilized TvDAO fusion proteins maintained 89 and 66% of initial activities. Therefore, the biomimetic silicification of TvDAO in form of the fusion protein with R4 or R5 peptide not only enhanced the pH and thermal stabilities, but also improved the oxidative tolerance toward H2O2.

並列關鍵字

SILAFFIN ； BIOMIMETIC SILICIFICATION ； D-AMINO ACID OXIDASE

參考文獻

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