PURIFICATION AND CHARACTERIZATION OF UPSILON-HEXACHLOROCYCLOHEXANE (HCH) DEHYDROCHLORINASE FROM BURKHOLDERIA PSEUDOMALLEI T4

摘要

Bacterial isolates belonging to HCH -degrading microbial consortium were screened individually for HCH - dehydrochlorinase activity. Among these, the cell free extract of Burkholderia pseudomallei T_4 showed higher UPSILON-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 °C and was stable for 2 h. The K_m and V_(max) 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 alpha-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.