PURIFICATION AND CHARACTERIZATION OF ϒ- HEXACHLOROCYCLOHEXANE (HCH) DEHYDROCHLORINASE FROM BURKHOLDERIA PSEUDOMALLEI T4I.M. MANDAPPA, H.M. RAJASHEKARA MURTHY, P.R. RAMASARMA, M.S. THAKUR AND H.K.MANONMANIAbstract Bacterial isolates belonging to HCH -degrading microbial consortium were screened individually for HCH - dehydrochlorinase activity. Among these, the cell free extract of Burkholderia pseudomallei T4 showed higher ϒ-HCH- dehydrochlorinase activity. The enzyme was purified to apparent homogeneity using Sephorose 6B gel permeation chromatography with 4.8 fold purification. The purity was confirmed using SDS - PAGE, capillary electrophoresis and HPLC. The enzyme had pH optimum of 6.0 and temperature optimum of 28 oC and was stable for 2 h. The Km and Vmax values of the enzyme were 2.162 and 39.52, respectively. Molecular weight of the enzyme was ∼32 KDa on SDS- PAGE. The enzyme was found to be a glycoprotein with mannose forming the carbohydrate backbone. The enzyme appeared to be a metallo protein containing Zn, Mg and Ca. The enzyme was inhibited by monovalent and divalent cations. The purified enzyme showed reactivity with all isomers of HCH except α-isomer and was not reactive against other halo aromatic derivatives. Serine and tryptophan residues were present in the active site. The N-terminal sequence was found to be AIGRVHNA.
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