Fig 1.
SDS-PAGE and gel-filtration chromatography of recombinant Nostoc sp.
9R-LOX. a SDS-PAGE analysis of each purification step. Lanes: 1, prestained marker protein (95, 72, 68, 55, 43, 34, 26 and 17 kDa); 2, control strain containing plasmid pET-15b without lox gene; 3, crude extract; 4, purified enzyme. b Gel-filtration chromatography. Ferritin (400 kDa), catalase (206 kDa), and conalbumin (75 kDa) (filled circle); and 9R-LOX (empty circle).
Fig 2.
Identification of reaction product of 9R-LOX by HPLC and LC-MS/MS.
The reaction was performed in 50 mM Tris-HCl buffer (pH 8.5) containing 10 mM ALA, 0.05% Tween 80, and 0.25 U ml−1 enzyme at 20°C for 10 min. a Reversed-phase HPLC for 9R-HPOTE and reduced 9R-HOTE formation from ALA. b Reversed-phase HPLC for 9R-HOTE formation and substrate (ALA) reduction. The control reaction was carried out without the addition of 9R-LOX. c LC-MS/MS of converted product by 9R-LOX. d Chiral-phase HPLC for 9R-HOTE.
Fig 3.
Effect of solvent on the activity of 9R-LOX from Nostoc sp.
The data represent the means of three separate experiments, and error bars represent the standard deviation. a Effect of solvent type. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA, 0.1 U ml−1 enzyme, and 4 (black bar) or 6% (grey bar) (v/v) of solvent at 15°C for 10 min. b Effect of acetone concentration. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA and 0.1 U ml−1 enzyme by varying the acetone concentration from 1 to 10% (v/v).
Fig 4.
Effect of detergent on the activity of 9R-LOX from Nostoc sp.
The data represent the means of three separate experiments, and error bars represent the standard deviation. a Effect of detergent type. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA, 0.1 U ml−1 enzyme, and 0.02 (black bar) or 0.1% (grey bar) (w/v) of detergent at 15°C for 10 min. b Effect of Tween 80 concentration. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA and 0.1 U m−1 enzyme by varying the Tween 80 concentration from 0.005 to 1% (w/v) at 15°C for 10 min.
Table 1.
Specific activity and kinetic parameters of recombinant Nostoc sp. 9R-LOX for PUFAs.
Fig 5.
Effects of the concentrations of enzyme and substrate on the production of 9R-HOTE from ALA by 9R-LOX.
The data represent the means of three separate experiments, and error bars represent the standard deviation. a Effect of enzyme concentration. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 10 g l−1 ALA, 5% (v/v) acetone, and 0.2% (w/v) Tween 80 at 15°C for 20 min by varying the enzyme concentration from 0.05 to 2 g l−1. b Effect of ALA concentrations. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.5 g l−1 enzyme, 5% (v/v) acetone, and 0.2% (w/v) Tween 80 at 15°C for 20 min by varying the ALA concentrations from 10 to 60 g l−1.
Fig 6.
Time-course reactions for the production of fatty acids by CRL, 9R-HOTE and 9R-HODE by recombinant 9R-LOX.
The data represent the means of three separate experiments, and error bars represent the standard deviation. a Production of fatty acids from PO by CRL. Fatty acids were α-linoleic acid (filled circle), linoleic acid (empty circle), oleic acid (filled inverse triangle), and other fatty acids (empty triangle). The reactions were performed in 50 mM Tris-HCl buffer (pH 7.5) containing 1 g l−1 CRL, 10 g l−1 PO, and 0.02% (w/v) Tween 80 at 37°C with agitation at 200 rpm for 2 h. b Production of 9R-HOTE (empty circle) and 9R-HODE (empty triangle) from ALA (filled circle) and LA (filled inverse triangle) in PO hydrolyzate. The reactions were performed in a 500 mL baffled flask in 80 mL of 50 mM Tris-HCl buffer (pH 8.5) consisting of PO hydrolyzate, which contained 40 g l−1 ALA, 10 g l−1 LA, 15.3 g l−1 of other fatty acids, 5% (v/v) acetone, 0.2% (v/v) Tween 80, and 1 g l−1 purified 9R-LOX at 15°C for 100 min with agitation at 250 rpm. c Production of 9R-HOTE (empty circle) from reagent grade ALA (filled circle) by 9R-LOX. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 40 g/L reagent-grade ALA, 1 g l−1 9R-LOX, 5% (v/v) acetone, and 0.2% (w/v) Tween 80 at 15°C for 80 min. d Production of 9R-HODE (empty inverse triangle) from reagent-grade LA (filled circle) by 9R-LOX. The reactions were performed in the same manner as those of 9R-HOTE production, except for the substrate LA.
Table 2.
Production of HPFA and HFA from PUFA and oil hydrolyzate by LOX.