Cloning and expression of lipoxygenase gene from Anabaena sp. PCC 7120 and purification, characterization of the recombinant enzyme.
- Author:
Chong ZHANG
1
;
Xiaowei ZHOU
;
Fengxia LÜ
;
Xiaomei BIE
;
Tingting TAO
;
Qi YING
;
Zhaoxin LU
Author Information
1. Laboratory of Enzyme Engineering, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Anabaena;
enzymology;
genetics;
Catalytic Domain;
Cloning, Molecular;
Enzyme Stability;
Escherichia coli;
metabolism;
Lipoxygenase;
chemistry;
genetics;
Metals, Heavy;
chemistry;
Mutagenesis, Site-Directed;
Recombinant Proteins;
chemistry;
genetics
- From:
Chinese Journal of Biotechnology
2012;28(4):440-456
- CountryChina
- Language:Chinese
-
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.
