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

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Title
Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H.
Other Titles
호냉성 박테리아 (Colwellia psychrerythraea 34H) 유래 저온 활성 lipase 단백질의 정제, 활성측정 및 구조해석을위한 X-선 회절 실험결과 분석
Authors
Do, Hackwon
Kim, Hak Jun
Lee, Sung Gu
Eom, Soo Hyun
An, Jun Yop
Song, Hye Eun
Kwon, Mi Hyun
Lee, Jun Hyuck
Subject
Biochemistry & Molecular Biology; Biophysics; Crystallography
Keywords
Colwellia psychrerythraea; CpsLip; cold adaptation; lipases; α/β-hydrolase fold
Issue Date
2013
Publisher
International Union of Crystallography
Citation
Do, Hackwon, et al. 2013. "Purification, characterization and preliminary X-ray diffraction analysis of a cold-active lipase (CpsLip) from the psychrophilic bacterium Colwellia psychrerythraea 34H.". ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 69(8): 920-924.
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, designated the UniProtKB code Q486T5, was expressed as an N-terminal hexa-histidine fusion protein in Escherichia coli and purified by affinity and size exclusion chromatography. The expression and purification of CpsLip enabled the characterization of the protein’s lipase enzymatic properties. The optimal activity temperature and pH of the recombinant protein were 25°C and pH 7, respectively. CpsLip maintained over 80% activity in the low temperature range (5? 15°C), thereby suggesting that CpsLip is a cold-active lipase. Substrate specificity analysis demonstrated that CpsLip exhibited the maximum activity toward the C12 acyl group. In addition, our sequence alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135, and His283. Moreover, the purified CpsLip was successfully crystallized using the hanging-drop vapor diffusion method, and a complete diffraction data set was collected to 4.0 ? resolution using a synchrotron X-ray radiation source on the BL-5A beamline of the Photon factory.e fusion protein in Escherichia coli and purified by affinity and size exclusion chromatography. The expression and purification of CpsLip enabled the characterization of the protein’s lipase enzymatic properties. The optimal activity temperature and pH of the recombinant protein were 25°C and pH 7, respectively. CpsLip maintained over 80% activity in the low temperature range (5? 15°C), thereby suggesting that CpsLip is a cold-active lipase. Substrate specificity analysis demonstrated that CpsLip exhibited the maximum activity toward the C12 acyl group. In addition, our sequence alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135, and His283. Moreover, the purified CpsLip was successfully crystallized using the hanging-drop vapor diffusion method, and a complete diffraction data set was collected to 4.0 ? resolution using a synchrotron X-ray radiation source on the BL-5A beamline of the Photon factory.
URI
http://repository.kopri.re.kr/handle/201206/5834
DOI
http://dx.doi.org/10.1107/S1744309113019428
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