Open AccessPurification, characterization and preliminary X‐ray diffraction analysis of a cold‐active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H
Author(s) -
Do Hackwon,
Lee Jun Hyuck,
Kwon Mi Hyun,
Song Hye Eun,
An Jun Yop,
Eom Soo Hyun,
Lee Sung Gu,
Kim Hak Jun
Publication year - 2013
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309113019428
Subject(s) - psychrophile , lipase , escherichia coli , enzyme , biochemistry , active site , chemistry , biology , chromatography , gene
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.