Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H

• Published in: Acta crystallographica. Section F, Structural biology and crystallization communications Structural biology and crystallization communications, 2013-08, Vol.69 (8), p.920-924
• Main Authors: Do, Hackwon, Lee, Jun Hyuck, Kwon, Mi Hyun, Song, Hye Eun, An, Jun Yop, Eom, Soo Hyun, Lee, Sung Gu, Kim, Hak Jun
• Format: Article
• Language: English
• Subjects: Alteromonadaceae - enzymology; Alteromonadaceae - genetics; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; cold adaptation; Cold Temperature; Colwellia; Colwellia psychrerythraea; CpsLip; Crystallization Communications; Diffraction; E coli; Escherichia coli; Lipase; Lipase - chemistry; Lipase - genetics; Lipase - isolation & purification; lipases; Low temperature; Manufacturing engineering; Metalloendopeptidases; Molecular Sequence Data; Photons; Proteins; Purification; Recombinant; X-Ray Diffraction; α/β-hydrolase fold
• ISSN: 1744-3091; 1744-3091; 2053-230X
• DOI: 10.1107/S1744309113019428

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.