- 學位論文
重組Haematonectria haematococca D型胺基酸氧化酶生化特性探討
Biochemical Characterization of Recombinant D-Amino Acid Oxidase from Haematonectria haematococca
摘要
由Haematonectria haematococca 94071401選殖出的DNA cDNA基因建構於表現載體pET23am後，置於E. coli BL-21(DE3)中進行異源表現。於25 oC下以含sorbitol與betaine的terrific broth進行培養時，經IPTG誘導後，於第16小時可獲得最高活性，約為105,000 U/L。以SDS PAGE分析清楚顯示，所表現的重組Haematonectria haematococca DAO (HhDAO)分子量約為41 kDa。利用HhDAO於C端具His-tag的特性，以金屬螯合層析法純化後，進行生化特性分析。HhDAO的最適反應溫度為25oC，TM為49oC；最適反應pH為8，而pH穩定性以pH 7時最佳。經10 mM H2O2 處理後的半衰期分別約為510分鐘，和Rhodosporidium toruloides DAO相似。20種D型胺基酸中， D-Trp為受質時有最佳kcat/KM值，約430.5 s-1 mM-1。以0.1 mg/ml的濃度儲存在4和25oC下30天，仍分別保有80和72%的殘餘活性。
並列摘要
The DAO cDNA gene cloned from Haematonectria haematococca 94071401 was constructed onto pET23am and transformed into E. coli for heterologous expression. The SDS PAGE analysis clearly showed that the molecular weight of the recombinant H. haematococca DAO (HhDAO) expressed was approximately 41 kDa as expected. When overexpressed in terrific broth containing sorbitol and betaine at 25oC, the HhDAO activities reached a maximum of 105,000 U/L 16 hr after IPTG induction. The recombinant HhDAO was purified through its C-terminal His tag by metal chelation chromatography for biochemical characterization. The optimal temperature and TM were 25 and 49oC, respectively. The pH optimum was 8 while the best pH stability was at pH 7. The half-life for the oxidative resistance to 10 mM H2O2 was 510 min, which is comparable to that for Rhodosporidium toruloides DAO. Among the 20 D-amino acids examined, HhDAO had a best kcat/KM value of 430.5 s-1 mM-1 toward D-Trp. After 30-day storage at 4 and 25oC, HhDAO with a concentration of 0.1 mg/ml remained 80 and 72% residual activity, respectively.