Open AccessScreening and characterization of trehalose‐oleate hydrolyzing lipase
Author(s) -
Ishimoto Ryo,
Sugimoto Manabu,
Kawai Fusako
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10526.x
Subject(s) - chromatography , lipase , size exclusion chromatography , chemistry , enzyme , enzyme assay , burkholderia , ultrafiltration (renal) , biochemistry , bacteria , biology , genetics
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 p I of the enzyme was 6.3. The enzyme showed the maximum activity at pH 9 and 65°C, and was stable in the range of pH 5–10 and up to 60°C. Almost all the activity (92%) was kept after incubation for more than 1 week at 50°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.