title: Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200.
abstract: Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5-12 and active at pH 5.5-6, showing optimal activity at pH 7.0 at 45 degrees C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5-20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.
authors.affiliation: Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho, 4259, Midori-ku, Yokohama, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Takeda
authors.forename: Rikizo
authors.initials: R
authors.lastname: Aono
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Doukyu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002787:Sterol Esterase