- 學位論文
耐輻射奇異球菌過氧化氫酶之選殖表現及生化研究
Cloning, expression, and biochemical studies of the catalase from Deinococcus radiodurans R1
摘要
中文摘要 耐輻射奇異球菌對於環境中的離子輻射、過氧化氫、紫外線及乾燥的抗性極高。耐輻射奇異球菌的細胞萃取中，已經發現有很強的過氧化氫酶活性，其活性的強度，會受到細胞中過氧化氫濃度而影響。另外，在培養耐輻射奇異球菌時，加入過氧化氫可以誘導過氧化氫酶的產生。所以將耐輻射奇異球菌的過氧化氫酶基因放大後選殖到載體pET-28a，利用大腸桿菌的表現系統大量表現。經過鎳親和性層析法純化後，獲得的重組蛋白質是在N端帶有His-tag的過氧化氫酶，DrCatA (D. radiodurans Catalase A)。得到約2 mg有活性的DrCatA，比活性約為83.97 U/mg。再以DEAE離子交換樹脂純化得到約0.4 mg有活性的DrCatA，比活性約為128.5 U/mg。最後以西方墨點法，確定DrCatA為帶有His-tag的重組蛋白質。再經由高效能液相層析法及質譜分析確定其分子量，質譜分析的實驗值比理論值少193 Da是目前還不知道的原因。 然而apo-DrCatA是需要輔酶hemin才能形成具有活性的holo-DrCatA。另外，在實驗上也發現hemin有過氧化氫酶的催化活性，具有分解過氧化氫的能力。在重組過氧化氫酶的酵素動力學研究中，它的最大催化速率為6.7 (μmol/ml min)；Km是10.1 (mM)；而其kcat (turnover number) 的值為42402.3 (min-1)，所以kcat/Km為2.53 × 108 (M-1sec-1)，其比活性則是250.9 (U/mg) 或15180.1 (U/μmol)。另外，在血基質的酵素動力學中，它的最大速率為2.7 (μmol/ml min)；Km是18.1 (mM)；而其kcat的值為54.6 (min-1)，所以kcat/Km為1.813 × 105 (M-1sec-1)，其比活性則是27.4 (U/mg) 或17.3 (U/μmol)。由以上實驗的Km值我們可以得知，重組過氧化氫酶與受質結合的親和力比血基質好；而從kcat/Km值顯示，在受質濃度飽和狀態下重組過氧化氫酶的催化效率比血基質多出一千倍。
並列摘要
Abstract Deinococcus radiodurans R1 is well-known for its extraordinary resistance against ionizing radiation, oxidative stress, UV light, heat, and desiccation. Strong catalase activities are found in D. radiodurans cell extracts, and the level of activity can be influenced by exposing cells to a sublethal dose of H2O2. The enzyme of catalase is induced when D. radiodurans cultures are pretreated with H2O2. The gene encoding catalase A from D. radiodurans was amplified and cloned into the vector pET-28a and expressed in E. coli BL21. The expressed DrCatA (Catalase A from D. radiodurans) was purified to apparent homogeneity by affinity chromatography on Ni-NTA column, obtaining 2 mg of purified protein with specific activity of 83.97 U/mg. And 0.4 mg protein is obtained with specific activity of 128.5 U/mg after DEAE ion exchange chromatography. We have used the western blotting to confirm that DrCatA is a His-tag recombinant protein. The molecular mass was analysed by mass spectrum after desalting with reverse-phase HPLC through a C8 column. However, the reason of the experimental molecular mass being less than the theoretical molecular mass of 193 Dalton still remains unknown. DrCatA, with its coenzyme hemin, is able to transform from an inactive apo-DrCatA into an active holo-DrCatA. Hemin by itself also has the catalatic activity, and it can dismutate the hydrogen peroxide into water and oxygen. In the recombinant DrCatA kinetics study, the Vmax is 6.7 μmol/ml min with Km and its turnover number, kcat, of 10.1 mM and 42402.3 min-1 respectively. And the kcat/Km is 2.53 × 108 M-1sec-1 with specific activity of 250.9 U/mg or 15180.1 U/μmol. On the part of hemin kinetics study, the Vmax is 2.7 μmol/ml min with Km and its turnover number, kcat, of 18.1 mM and 54.6 min-1 respectively. And the kcat/Km is 1.813 × 105 M-1sec-1 with specific activity of 27.4 U/mg or 17.3 U/μmol. DrCatA had a greater affinity for the substrate than hemin as determined by their Michaelis constant, Km. As a result from their kcat/Km values, the catalytic efficiency of DrCatA is approximately 103 folds greater than that of hemin operating at substrate concentrations substantially below saturation amounts.
參考文獻
延伸閱讀
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