| Summary: | 碩士 === 國立東華大學 === 物理學系 === 102 === Lysine 5,6-Aminomutase (5,6-LAM) is one of the coenzyme B12-dependent enzyme. 5,6-LAM is an α 2 β 2 tetramer that can be thought of as a dimer of αβ units. The molecular weights of the α and β subunits are 55 and 30 kDa respectively, and the total molecular weights of 5,6-LAM is approximately 170 kDa.
We used the purification of 5,6-LAM with hydrophobic chromatography, and finally stored in the Distilled Deionized water, but we could not attain spectroscopic evidence for radical intermediate in the reaction of 5,6-LAM by electron paramagnetic resonance (EPR). And we found less dissolved oxygen solubility and may stabilize the folded form of the protein at pH 7.0 potassium phosphate by references, therefore we attempts to change the buffer solution of the hydrophobic chromatography to be improved.
In addition, we performed the purification of 5,6-LAM with high activity but has impurity problems by the salinity concentration gradient. we solve this problem with changing the salinity concentration gradient when purification with FPLC column.
Finally, we set up another method of purification system. QFF column change to DE52 column, and change the salinity concentration gradient and buffer. 5,6-LAM also can get the same EPR signal, and has better concentration and volume than before. It is a good way from phenyl column to DE52 column for purification.
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