390212 Heterogeneous Expression, Biochemical Characterization, and Thermostabilization of Pseudomonas Aeruginosa Lipoxygenase

Wednesday, November 19, 2014
Galleria Exhibit Hall (Hilton Atlanta)
Song Liu, Xinyao Lu, Jian Chen and Guocheng Du, School of Biotechnology, Jiangnan University, Wuxi, China

Lipoxygenase (LOX; EC is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this presentation, we described the heterogeneous expression, characterization, and thermostabilization of Pseudomonas aeruginosa LOX.

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 Km and Vmax 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.

Two highly flexible loops (an N-terminal loop and an internal loop) was modified to improve the thermal stability of LOX. Deletion of the first 20 and 30 residues of the N-terminal loop increased the thermal stability of the LOX by 1.3 and 2.1 fold, respectively. Although deletion of the internal loop led to a sharp reduction of both thermal stability and catalytic activity, the residue substitutions with the glycines (G204P, G206P, and G204P/G206P) or even glycine-rich linker (L6/PT) in this internal loop increased the thermal stability of the LOX by values ranging from 0.46 to 3.45 fold. Over 85% of the specific activity was maintained in all thermally stabilized LOX mutants. Circular dichroism analysis showed that the α-helix and β-sheet content was not changed by the loop modifications. Fluorescence spectra analysis indicated that the thermally stabilized mutants exhibited a more compact structure.

To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms and increasing the thermal stability of LOX by protein engineering without remarkably affecting the catalytic rate.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division