Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli
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- 作者：Xinyao Lu (1) (3)
Juan Zhang (1) (3)
Song Liu (1) (3)
Dongxu Zhang (1) (3)
Zhi Xu (1) (3)
Jing Wu (3) (4)
Jianghua Li (1) (3)
Guocheng Du (1) (3)
Jian Chen (2) (3) - 关键词：Lipoxygenase ; Secretory expression ; Escherichia coli ; Catalytic properties
- 刊名：Applied Microbiology and Biotechnology
- 出版年：2013
- 出版时间：July 2013
- 年：2013
- 卷：97
- 期：13
- 页码：5793-5800
- 全文大小：314KB
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Juan Zhang (1) (3)
Song Liu (1) (3)
Dongxu Zhang (1) (3)
Zhi Xu (1) (3)
Jing Wu (3) (4)
Jianghua Li (1) (3)
Guocheng Du (1) (3)
Jian Chen (2) (3)
1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China
3. School of Biotechnology, Jiangnan University, Wuxi, 214122, China
4. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China
2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China - ISSN：1432-0614
文摘
Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1?mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20?°C for 47?h, the titer of the recombinant enzyme reached 3.89?U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70?kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K m and V max of the recombinant enzyme were 48.9?μM and 0.226?μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25?°C and pH?7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.