1. Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H.
- Author
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Do H, Lee JH, Kwon MH, Song HE, An JY, Eom SH, Lee SG, and Kim HJ
- Subjects
- Alteromonadaceae genetics, Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins isolation & purification, Lipase genetics, Lipase isolation & purification, Metalloendopeptidases, Molecular Sequence Data, X-Ray Diffraction, Alteromonadaceae enzymology, Bacterial Proteins chemistry, Cold Temperature, Lipase chemistry
- 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.
- Published
- 2013
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