Purification and Function analysis of the broad-spectrumorganomercurial lyase protein ( MerB3 ) from mercury resistance transposon, TnMERI1

• Main Author: 林慧姿
• Other Authors: 黃介辰
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/48399281968352370190

碩士 === 國立中興大學 === 生命科學系所 === 96 === Organomercury, in particular, are potent neurotoxuns because they bind so strongly to protein residue and are capable of crossing the blood-brain barrier. Detoxification of organomercury compounds is therefore of critical importance. In nature, some bacterial strains have occurred mercury resistance . Bacterial organomercurial lyase(MerB) conducts protolytic cleavage of the Hg-C bond while another enzyme, mercuric ion reductase(MerA), reduces Hg(II) to less toxic elemental mercury, Hg(0). From Minamata Bay isolated B. megaterium MB1, three merB genes(merB1, merB2, merB3) were found encoded in the same transposon TnMERI1. While MerB3 has been found that it performs a broader substrate specificity than others. In this study, the MerB3 protein was purified in to homogeneity with three kinds of columns. It catalyzes the protonolysis of the C-Hg bond in a wide range of organomercurial complex (alkyl, aryl), and shows more broad substrate specificity than MerB2. We have found that the reaction works good under the existence of thiol group(DTT, dithiothreitol). This may due to the thiol group facilitates demercuration of the enzyme-Hg2+ complex after the cleavage of the C-Hg bond. The Kinetic parameters of MerB3 was obtained from this study, while the Kcat for MerB3 with PMA(Mercury phenyl acetate) is 2x10-2 min -1 and K m is 4.5x10-3mM. On the other hand, the Kcat value of another substrate PHMB (p-Chloromercuribenzoate) is 9.1x10-4 min-1and Km is 7.5x10-3mM.