Isolation of a Novel Lipase Gene from <Emphasis Type="Italic">Serratia liquefaciens</Emphasis> S33 DB-1, Functional Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its Properties |
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Authors: | Hongyan Yao Shunwu Yu Lida Zhang Kaijing Zuo Hua Ling Fei Zhang Kexuan Tang |
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Institution: | (1) Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, School of Life Science and Technology, Shanghai Jiao Tong University, Shanghai, 200030, P.R. China;(2) Shanghai Agrobiological Gene Center, Shanghai Academy of Agricultural Sciences, Shanghai, 201106, P.R. China;(3) State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai, 200433, P.R. China |
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Abstract: | A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino
acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host,
methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose
plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase
activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml−1. For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS. |
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Keywords: | Cloning Expression Genomic-walking Lipase Pichia pastoris Serratia liquefaciens |
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