- 學位論文
共同表現融合透明顫菌血紅蛋白對重組Rhodosporidium toruloides D型胺基酸氧化酶於大腸桿菌中生產的影響
The Effect of Co-expression of Double Vitreoscilla Hemoglobin on Production of Recombinant Rhodosporidium toruloides D-Amino Acid Oxidase in E. coli
摘要
本研究的主要目的是將能攜帶氧分子的透明顫菌血紅蛋白的兩個相同的對應基因，藉由遺傳工程技術將其銜接融合，並以S10 promotor驅動表現產生融合血紅蛋白質- double Vitreoscilla hemoglobin (double VHb)，而且探討其對D型胺基酸氧化酶於大腸桿菌中異源表現的影響。當有double VHb共同表現時，在LB broth和TB培養液中，DAO的活性較無double VHb共同表現時，大約分別提升了1.16倍和1.12倍。 當菌液濃度達到OD600=1.5時，若分別加入0.1、0.2、0.3和0.4 mM IPTG誘導，則於LB broth中，由pDAO表現的DAO活性以0.3 mM IPTG誘導的38.6 U/ml為最高，而由pSdVD表現的活性則以0.3 mM IPTG誘導的43.4 U/ml為最高。而於TB broth中，由pDAO表現的DAO活性則以0.4 mM IPTG誘導的28.7 U/ml為最高。由pSdVD表現的DAO活性以0.1 mM IPTG誘導的40.7 U/ml為最高。
並列摘要
The main goal of this study is to perform the gene fusion of Vitreoscilla hemoglobin (VHb), a oxygen-binding homodimeric protein, to produce double VHb with two subunits covalently linked together and to examine the expression level of D-amino acid oxidase in E. coli with or without the co-expression of double VHb driven by S10 promoter. In the presence of double VHb, the DAO activities in LB broth and TB were increased by 1.16 and 1.12-fold, respectively, as compared with those with the DAO expression only. When induced by 0.1, 0.2, 0.3 and 0.4 mM IPTG at OD600=1.5, the maximal DAO activity, 38.6 U/ml, expressed from pDAO in LB broth was observed with addition of 0.3 mM IPTG. The maximal activity from pSdVD was 43.4 U/ml obtained under the same induction condition. Using TB as the culture medium, the maximal DAO activities from pDAO and pSdVD were 28.7 and 40.7 U/ml, respectively, as induced with addition of 0.4 and 0.1 mM IPTG, respectively.