Cloning, expression and characterization of esterase from Pseudomonas putida 3SK = Pseudomonas putida 3SK esterase의 유전자 cloning과 발현 및 특성 연구

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Pseudomonas putida 3SK, a solvent resistant strain, produced extracellular lipase. We observed that there was another lipolytic enzyme, which had no lipase activity in P. putida 3SK. This lipolytic enzyme, an esterase, was cloned in Escherichia coli and sequenced. The cloned esterase gene, which designated estA, had single open reading frame of 1941 nucleotides. The estA gene encoded a protein of 646 amino acids and was predicted to belong to the GDSL family of lipolytic enzymes. The consensus motif GxSxxDxG, which was a characteristic feature of the GDSL lipolytic enzymes, was found in estA. A putative catalytic triad, $Ser^{38}$, $Asp^{172}$, and $His^{313}$ was also determined. The calculated molecular weight was 69.6kDa and theoretical isoelectric point (pI) was 4.68. The esterase expressed in E. coli was purified by Ni-NTA affinity chromatography and FPLC gel filtration chromatography. The molecular weight of the purified esterase was about 65kDa and judging from the immunoblot analysis, an esterase from P. putida 3SK was extracellular enzyme. The optimum pH and temperature were pH 11 and 50℃, respectively. In the study of effect of organic solvents, DMF was the most effective solvent among the solvents tested for the p-nitrophenyl myristate hydrolysis. On the basis of substrate specificity and sensitivity toward various inhibitors, P. putida 3SK esterase appears to be an acetylesterase (E.C. 3.1.1.6).
Advisors
Rhee, Joon-Shick이준식
Description
한국과학기술원 : 생물과학과,
Publisher
한국과학기술원
Issue Date
1999
Identifier
151576/325007 / 000973392
Language
eng
Description

학위논문(석사) - 한국과학기술원 : 생물과학과, 1999.2, [ vi, 79 p. ]

Keywords

Pseudomonas putida; Esterase; GDSL lipolytic enzyme; GDSL 지질분해 효소; 슈도모나스 퓨티다; 에스테르가수분해효소

URI
http://hdl.handle.net/10203/28584
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=151576&flag=dissertation
Appears in Collection
BS-Theses_Master(석사논문)
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