Screening and characterization of trehalose-oleate hydrolyzing lipase.
Various soil samples were collected to screen the presence of microorganisms which have ability to degrade TOE. One strain (AKU-883) with good TOE degrading activity was isolated and identified as Burkholderia cepacia and the extracellular enzyme was purified to homogeneity. The purification was achieved by ultrafiltration, Super Q anion-exchange chromatography and Superdex 200HR gel-filtration in the presence of Triton X. The enzyme was purified to 85-fold, and specific activity of 4.910 kU mg protein(-1). The peak preparation on gel filtration showed a single band of 34 kDa on SDS-PAGE and native PAGE which indicate the monomeric nature of the enzyme. The pI of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65 degrees C, and was stable in the range of pH 5--10 and up to 60 degrees C. Almost all the activity (92%) was kept after incubation for more than 1 week at 50 degrees C (pH 7.3). High activities remained even in water-miscible solvents such as ethanol, dimethyl formamide, diisopropyl ether, and dioxane. The N-terminal 16 amino acid residues were determined as A-N-G-Y-A-A-T-R-Y-P-I-I-L-V-G-G, which showed a consensus sequence for lipases from Burkholderia species. Thus the enzyme was concluded to be a kind of lipase.
Ishimoto
R
R
Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki 710-0046, Japan. ishimoto@rib.okayama-u.ac.jp
Sugimoto
M
M
Kawai
F
F
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D002384:Catalysis; D004795:Enzyme Stability / Q000187:drug effects; D004952:Esters; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism*; D012988:Soil Microbiology; D012997:Solvents / Q000494:pharmacology; D013696:Temperature; D014199:Trehalose / Q000378:metabolism
D004952:Esters; D012997:Solvents; D019301:Oleic Acid; D014199:Trehalose; D008049:Lipase