title: Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable lipase from Bacillus stearothermophilus P1.
abstract: The gene encoding a thermostable lipase secreted by Bacillus stearothermophilus P1 has been cloned and overexpressed in Escherichia coli. The recombinant lipase was purified to homogeneity using ammonium sulfate precipitation, anion-exchange chromatography (Poros 20 HQ) and Sephacryl S-200HR. The molecular mass was shown to be 43 209 Da by mass spectrometry. Crystals suitable for X-ray diffraction analysis were obtained by the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitating agent. Determination of the structure by molecular replacement with existing mesophilic lipase structures has proved unrewarding, as there is less than 20% sequence identity with known lipase structures, but preliminary results with heavy-atom soaking indicate that this strategy will allow the structure to be solved. The availability of this new lipase structure will be of particular significance because it will be the first thermostable lipase to be described.
authors.affiliation: Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
authors.forename: Supachok
authors.initials: S
authors.lastname: Sinchaikul
authors.forename: Joel D A
authors.initials: JD
authors.lastname: Tyndall
authors.forename: Linda A
authors.initials: LA
authors.lastname: Fothergill-Gilmore
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.forename: Suree
authors.initials: S
authors.lastname: Phutrakul
authors.forename: Shui Tein
authors.initials: ST
authors.lastname: Chen
authors.forename: Malcolm D
authors.initials: MD
authors.lastname: Walkinshaw
mesh_terms: D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D008025:Ligases