Cloning, expression, and biochemical studies of the catalase from Deinococcus radiodurans R1
Date Issued
2006
Date
2006
Author(s)
Lin, Wei-Ling
DOI
zh-TW
Abstract
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.
Subjects
耐輻射奇異球菌
過氧化氫酶
Deinococcus radiodurans R1
catalase
Type
other
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