Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinant enzyme / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 440-456, 2012.
Artículo
en Chino
| WPRIM
| ID: wpr-342472
ABSTRACT
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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Estabilidad de Enzimas
/
Proteínas Recombinantes
/
Química
/
Mutagénesis Sitio-Dirigida
/
Clonación Molecular
/
Anabaena
/
Lipooxigenasa
/
Metales Pesados
/
Dominio Catalítico
/
Escherichia coli
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2012
Tipo del documento:
Artículo
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