title: Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H.
abstract: The putative lipase CpsLip from the psychrophilic bacterium Colwellia psychrerythraea 34H encodes a 34,538 Da, 308-amino-acid protein. In this study, CpsLip (UniProtKB code Q486T5) was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli and purified by affinity and size-exclusion chromatography. The expression and purification of CpsLip enabled characterization of the lipase enzymatic properties of the protein. The optimal activity temperature and pH of the recombinant protein were 298 K and pH 7, respectively. CpsLip maintained over 80% activity in the low-temperature range (278-288 K), thereby suggesting that CpsLip is a cold-active lipase. Substrate-specificity analysis demonstrated that CpsLip exhibits maximum activity towards the C12 acyl group. In addition, sequence-alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135 and His283. Moreover, purified CpsLip was successfully crystallized using the hanging-drop vapour-diffusion method and a complete diffraction data set was collected to 4.0 Å resolution using synchrotron radiation on the BL-5A beamline of the Photon Factory.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.forename: Mi Hyun
authors.initials: MH
authors.lastname: Kwon
authors.forename: Hye Eun
authors.initials: HE
authors.lastname: Song
authors.forename: Jun Yop
authors.initials: JY
authors.lastname: An
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: Sung Gu
authors.initials: SG
authors.lastname: Lee
authors.forename: Hak Jun
authors.initials: HJ
authors.lastname: Kim
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003080:Cold Temperature; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D008049:Lipase; D008666:Metalloendopeptidases; C110245:cold alkaline protease
keywords: Colwellia psychrerythraea; CpsLip; cold adaptation; lipases; α/β-hydrolase fold
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