Cloning and expression of a lipase gene from pseudomonas sp. in E. coli = Pseudomonas sp.로부터 Lipase 유전자의 cloning 및 대장균에서의 발현

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A lipase gene (lipK) from Pseudomonas sp. KFCC 10818 was cloned in Escherichia coli JM83. The nucleotide sequence revealed the presence of two open reading frames of 933 and 492 nucleotides, respectively. The lipK gene was assumed to encode a lipase of311 amino acids. The gene was used GTG as initiation codon instead of ATG. The lipK gene alone was not conferred a lipase phenotype when expressed in E. coli using the tac promoter of the pKK223-3 plasmid. Downstream from lipK an extra open reading frame was identified and named limK. The limK gene could presumably encode a protein of 164 amino acids. The lipK was well expressed only in the presence of the complete limK. Some lipase genes from Pseudomonas have lipase expression-regulating gene. The protein from this gene has not been isolated or characterized yet. The predicted amino acid sequence of the lipase was shown 36-54\% identity to the modulator- or activator-containing lipases of Pseudomonas species. But no protein homologous to LimK, limK gene product, was identified. A putative catalytic triad, $Ser^{108},\; Asp^{255},\; His^{277}$, was determined by the comparison of the amino acid sequence of the lipase from Pseudomonads and threedimensional structure of Pseudomonas glumae. Extremely high identity was observed in the regions containing the consensus sequence of lipase, Gly-X-Ser-X-Gly. Lipase production in E. coli JM109 containing both lipK and limK gene started in the early exponential phase of cell growth. Most of the lipase activity was found in the cytoplasm. The cell extract was applied on ion exchange column, hydrophobic interaction column, and gel filtration column. The final product was 35-fold purified with a yield of 17\%. The optimum pH and temperature of the lipase was pH 9.0 and $70^\circ C$ (in range of $20^\circ C$ to $70^\circ C$), respectively.
Yoo, Ook-Joonresearcher유욱준researcher
한국과학기술원 : 생명과학과,
Issue Date
98683/325007 / 000933098

학위논문(석사) - 한국과학기술원 : 생명과학과, 1995.2, [ v, 72 p. ]

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