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KMID : 0364820160520020192
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Korean Journal of Microbiology
2016 Volume.52 No. 2 p.192 ~ p.201
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A novel cold-active lipase from Psychrobacter sp. ArcL13: gene identification, expression in E. coli, refolding, and characterization
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Koo Bon-Hun
Moon Byung-Hern
Shin Jong-Suh
Yim Joung-Han
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Abstract
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Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi
Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of
recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84?90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl
decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at 40°C and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at 10°C and 20°C, respectively, compared to its maximal activity at 40°C.
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KEYWORD
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Psychrobacter sp. ArcL13, cold-active lipase, gene prospecting, refolding
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