tailieunhanh - Báo cáo khoa học: Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis

A novel long-chain acyl-CoA thioesterase fromAlcaligenes faecalishas been isolated and characterized. The protein was extracted from the cells with 1mNaCl, which required , single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homo-meric aggregates, of mean diameter nm, consisting of 22-kDa sub-units. | iFEBS Journal Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis Puja Shahi Ish Kumar Ritu Sharma Shefali Sanger and Ravinder S. Jolly Institute of MicrobialTechnology Chandigarh India Keywords Alcaligenes faecalis immunogold electron microscopy long-chain acyl-CoA p-nitrophenylesters thioesterase Correspondence R. S. Jolly Institute of Microbial Technology Sector 39 Chandigarh 160 036 India Fax 91 172 269 0585 Tel 91 172 269 0908 E-mail jolly@ These authors contributed equally to this paper tPresent address Department of Physiology and Biophysics University of Iowa Iowa city IA 52242 USA Present address Department of Chemistry Wesleyan University Middletown CT 06459 USA Received 19 January 2006 revised 12 March 2006 accepted 22 March 2006 doi A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 M NaCl which required single-step purification to yield near-homogeneous preparations. In solution the protein exists as homomeric aggregates of mean diameter nm consisting of 22-kDa subunits. MS MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of pmol-min-1 mg protein -1 and M respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 C and pH and varied between 60 and 80 at temperatures of 25-70 C and pH . The