- 學位論文
重組斑馬魚和Haematonectria haematococca D型胺基酸氧化酶的生化特性探討
BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT D-AMINO ACID OXDIASE FROM ZEBRAFISH AND HAEMATONECTRIA HAEMATOCOCCA
摘要
D型胺基酸氧化酶 (D-amino acid oxidase; DAO)是普遍存在於真核生物的一種黃酮類酵素，能催化D型胺基酸的氧化脫氨作用，形成對應的酮酸和氨，並同時產生過氧化氫。目前已被廣泛運用於工業和生物科技上，作為生物催化劑。近年更發現其在動物體中扮演重要的生理角色。在本研究中，將斑馬魚與Haematonectria haematococca 9404010、94071401和96030313 DAO (HhDAO 01、 02及03) 之cDNA基因轉殖入大腸桿菌中，使其進行異源表現，並經純化後探討其生化特性。SDS-PAGE分析清楚顯示，表現的重組斑馬魚DAO (zDAO) 和HhDAO的分子量皆如預期約40 kD。當以含betain和sorbitol的terrific borth培養時，最高可溶性zDAO以及HhDAO01、02和03表現量分別約1800、26000、37000和31000 U/L。zDAO與HhDAO的最適反應溫度分別是50和40℃，而TM值 (1小時處理後，活性剩餘50%時的溫度) 則分別是44與52℃。最適反應pH分別是pH 9.5 和8.5，而於pH 8.0有最佳pH穩定性。當zDAO以及HhDAO01、 02和03濃度為5 ug/ml時，對於10 mM過氧化氫的耐受半衰期分別約為77、288、289和199分鐘。zDAO的最佳受質為D-Met其kcat/KM值約23.8 s-1mM-1 ，而HhDAO01、02和03則對D-Trp具有最佳催化效率，依次分別為 81.7、112.5與 71.3 s-1mM-1 。
並列摘要
D-Amino acid oxidase (DAO) is a flavoenzyme ubiquitously present in eukaryotes. It can catalyze the oxidative deamination of D-amino acids to produce the corresponding keto acids, NH3 and H2O2. Besides being well-known as industrial biocatalyst, this enzyme has been proposed to have novel physiological functions in animals. In this study, we traneformed and overexpressed DAO cDNA genes from zebrafish、Haematonectria haematococca 9404010、94071401and 96030313 (Hh01, 02 and 03) into E. coli, and characterized their catalytic properties after purification. The SDS PAGE analysis clearly showed that the molecular weights of the recombinant zebrafish DAO (zDAO) and H. haematococca DAO (HhDAO) expressed were approximately 40 kDa as expected. When cultured in terrific broth containing betaine and sorbitol, the maximal expression of soluble zDAO as well as HhDAO01, 02 and 03 reached approximately 1800, 26000, 37000 and 31000 U/L, respectively. The optimal temperature of zDAO and HhDAO were 50 and 40℃, respectively, and the TM value (the temperature at which 50% activity is remained after 1 h incubation) were 44 and 52℃, respectively. Their pH optima were 9.5 and 8.5, respectively, while the best pH stability both appeared at pH 8. The half-lives of zDAO as well as HhDAO01,02 and 03 HhDAO for the oxidative resistance to 10 mM H2O2 at the concentrations of 5 ug/ml were 77, 288、289 and 199 min, respectively. The best substrate for zDAO was D-Met with a kcat/KM value of 23.8 s-1 mM-1, while HhDAO 01、02 and 03 exhibited the best catalytic efficiency toward D-Trp of 81.7、112.5 and 71.3 s-1 mM-1, respectively.