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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2016, Vol. 36 Issue (3): 43-52    DOI: DOI:10.13523/j.cb.20160307
    
Cloning, Expression and Characterization of a Novel Psychrophile Lipase from the Deep Sea of the South China Sea
CAO Ying-ying1,2, DENG Dun1,2, ZHANG Yun1, SUN Ai-jun1, XIA Fang-liang3, HU Yun-feng1
1. Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
2. University of Chinese Academy of Sciences, Beijing 100049, China;
3. Binzhou Food and Drug Inspection and Testing Center, Binzhou 256618, China
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Abstract  

A gene lipaseB5 encoding a lipase, was cloned from the genome of Pseudonocardia antitumoralis SCSIO 01299 isolated from the deep sea of the South China Sea. LipaseB5 contains 972bp and encodes a putative lipase of 323 amino acids. LipaseB5 exhibits 98% similarity with a putative lipase from Pseudonocardia sp. HH130629-09. LipaseB5 was cloned into expression vector pET28a(+), expressed in E.coli BL21(DE3) and further purified using Ni-NTA affinity chromatography. The optimal substrate tested of lipaseB5 was p-NPD, the optimal working temperature was 30℃, the optimal working pH was 7.5. Vmax and Km were 109.8μmol/min and 0.976mmol/L, respectively. The hydrolysis activity of lipaseB5 toward olive oil was 32.019 7U/mg. LipaseB5 could remain high activities under pH 7.5~8.5 and 10~20℃. LipaseB5 was thermal-stable at 10~40℃. Multiple metal ions could not affect the activities of LipaseB5 and low-concentration Li+ and Mg2+ could stimulate the activity of LipaseB5. LipaseB5 could stand high concentration of NaCl and could still remain 103% of its original activity in the presence of 100mmol/L NaCl. LipaseB5 could also stand many short-chain alcohols and surfactants. TritonX-100, Tween-80 and Tween-20 could even stimulate the activities of LipaseB5.

Key words： Resistance to surfactants      Resistance to organic solvents      Halotolerant enzyme      Deep-sea psychrophile lipase      Resistance to metal ions     
Received: 27 October 2015      Published: 14 December 2015
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CAO Ying-ying
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CAO Ying-ying, DENG Dun, ZHANG Yun, SUN Ai-jun, XIA Fang-liang, HU Yun-feng. Cloning, Expression and Characterization of a Novel Psychrophile Lipase from the Deep Sea of the South China Sea. China Biotechnology, 2016, 36(3): 43-52.

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https://manu60.magtech.com.cn/biotech/DOI:10.13523/j.cb.20160307     OR     https://manu60.magtech.com.cn/biotech/Y2016/V36/I3/43

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