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We cloned the lipoxygenase gene (ana-LOX) from Anabaena sp. PCC 7120 and expressed it in Escherichia coli BL21 (DE3) pLysS. We determined the active site of the recombinant ana-LOX through site-directed gene mutagenesis and obtained the shortest length of the functional gene. Meanwhile, we studied the properties of recombinant ana-LOX after purification. The C-terminal of the Aos (allene oxide synthase)-LOX fusion gene in Anabaena sp. PCC 7120 genome was found belonging to LOXs family by bioinformatics analysis. Further results of site-directed gene mutagenesis confirmed that the active sites of ana-LOX were His197, His202, His369, Asn373and Ile455. The shortest length of functional gene was identified to be 1 254 bp based on the strategy of shortening the gene length gradually. The highest activity of recombinant ana-LOX of 6 750 U/mL could be achieved when constructed to pET-32a vector and expressed at low temperature 16 degrees C. We purified the enzyme by Ni-NTA chelating affinity chromatography, with 60.89% yield and specific activity of 11.4 x 10(4) U/mg. The optimum reaction temperature and pH for ana-LOX were 45 degrees C and 6.0, respectively. Furthermore, the obtained ana-LOX was stable at room temperature. The effect of metal ions on ana-LOX was determined also. Fe2+, Mg2+ Ca2+ could markedly promote the activity of this enzyme whereas Fe3+ and Cu2+ had a strong inhibitory effect on it. Finally, the ana-LOX could improve the microscopical structure of dough. The results of this study will provide a basis for future improvements and food industrial applications of ana-LOX.

Inactivation effect of pulsed electric field (PEF) on polyphenol oxidase (PPO) and lipoxygenase (LOX) was investigated using a laboratory PEF system with a coaxial treatment chamber. Circular dichroism (CD) and fluorescence analysis were used to study the conformation change of the protein. The experimental results show that PPO and LOX can be effectively inactivated by the PEF treatment. Inactivation effect of PPO and LOX increases with the increase in the applied electric strength and the treatment time. Activity of PPO and LOX can be reduced by 60.3% and 21.7% at 20 kV x cm(-1) after being treated for 320 micros respectively. The decrease of the negative peaks (208 and 215 nm in PPO spectra, 208 nm and 218 nm in LOX spectra) in CD spectra of PPO and LOX shows that PEF treatment caused a loss of alpha-helix and increase in beta-sheet content, indicating that conformation changes occur in the secondary structure of PPO and LOX enzyme. This effect was strengthened as the applied electric field increased: alpha-helical content of PPO and LOX was 56% and 29% after being treated at 8 kV x cm(-1), however, when the electric field was increased up to 20 kV x cm(-1), alpha-helical content of PPO and LOX decreased to 21% and 16% respectively. The decrease rate of alpha-helix and increase rate of beta-sheet in PPO are higher than LOX, indicating that the second conformation of PPO is less resistant to PEF treatment than LOX. The fluorescence intensity of LOX increases after PEF treatment. At the same time, increasing the applied pulsed electric field increases the fluorescence intensity emitted. Fluorescence measurements confirm that tertiary conformation changes occur in the local structure of LOX. However the possible mechanism of the conformation change induced by the PEF treatment is beyond the scope of the present investigation.

The effect of pulsed electric field (PEF) on the secondary and tertiary structure of lipoxygenase (LOX) in a buffer solution was analyzed using far UV-circular dichroism (CD) and fluorescence spectrophotometry, respectively. The secondary and tertiary structure of LOX changed after PEF treatment. The CD spectra of LOX also changed, with the intensity of two negative peaks and the content of alpha-helix significantly decreased (p < 0.05). The decrease in alpha-helix content in LOX showed a good linear correlation with the electric field strength. The fluorescence intensity of LOX increased, and the relative fluorescence intensity of the two characteristic peaks of LOX emission spectra at 337 nm and 583 nm also showed a good linear correlation with the electric field strength. These results showed that the activity inactivation of LOX may be due to the alteration in secondary structure, and both had a good relation.