Fig 1.
(A) SDS-PAGE of DAEase from F. plautii. Lanes: 1, pellet of C. glutamicum expressing DAEase; 2, crude extract of C. glutamicum expressing DAEase; 3, protein marker (130, 100, 75, 55, 40, 35, 25, and 17 kDa); 4, pellet of E. coli expressing DAEase; 5, crude extract of E. coli expressing DAEase; and 6, purified enzyme of E. coli expressing DAEase. (B) Determination of total molecular mass for DAEase from F. plautii by gel filtration chromatography with reference proteins. Ferritin (440 kDa), catalase (206 kDa), aldolase (158 kDa), and conalbumin (75 kDa); and DAEase from F. plautii.
Table 1.
Biochemical properties of DAEases and DTEases.
Table 2.
Substrate specificity of F. plautii DAEase for hexoketose.
Fig 2.
Effect of antibiotic treatment on the permeabilization of C. glutamicum expressing DAEase from F. plautii for the production of d-allulose from d-fructose.
(A) Effect of antibiotic treatment. 0 (white bar with diagonal line), 2 (black bar), 5 (white bar), 50 (gray bar), and 100 mg/L (gray bar with diagonal line). (B) Effect of penicillin concentration. The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 7.5 g/L cells and 50 mM d-fructose at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 3.
Effect of detergent treatment on the permeabilization of C. glutamicum expressing DAEase from F. plautii for the production of d-allulose from d-fructose.
(A) Effects of detergent treatment. 0 (white bar with dotted line), 0.2 (black bar), 0.5 (white bar), 2 (gray bar), and 5% (w/v) (gray bar with diagonal line). (B) Effect of span 20 concentration. The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 7.5 g/L cells and 50 mM d-fructose at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 4.
Effect of solvent treatment on the permeabilization of C. glutamicum expressing the DAEase from F. plautii for the production of d-allulose from d-fructose.
(A) Effect of solvent treatment. 0 (white bar with dotted line), 10 (black bar), and 20% (v/v) (white bar). (B) Effect of toluene concentration. The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 7.5 g/L cells and 50 mM d-fructose at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 5.
Effect of the combined treatment of permeabilizers on the permeabilization of C. glutamicum expressing DAEase from F. plautii for the production of d-allulose from d-fructose.
The concentrations of penicillin, span 20, and toluene used were 20 mg/L, 2% (w/v), and 10% (v/v), respectively. The reactions were performed in 50 mM PIPES buffer (pH 7.0) containing 7.5 g/L cells and 50 mM d-fructose at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 6.
Effect of metal ions on the production of d-fructose to d-allulose by nonpermeabilized and permeabilized cells of C. glutamicum expressing DAEase from F. plautii.
Production of d-allulose from d-fructose by nonpermeabilized cells (black bar) and permeabilized cells (white bar). The reactions were conducted in 50 mM PIPES buffer (pH 7.0) containing 7.5 g/L cells and 50 mM d-fructose in the presence of 1 mM metal ions at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 7.
Thermal stability of the activities of DAEase from F. plautii and DAEase from F. plautii in recombinant C. glutamicum cells.
(A) Thermal stability of the activity of recombinant cells. The permeabilized cells were incubated at 45°C (filled triangle), 50°C (empty square), 55°C (filled square), 60°C (empty circle), and 65°C (filled circle) for various incubation times. (B) Thermal stability of enzyme activity. The enzymes were incubated at 45°C (filled triangle), 50°C (empty square), 55°C (filled square), 60°C (empty circle), and 65°C (filled circle) for various incubation times. A sample was withdrawn at each time point and the relative activity was measured. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 8.
Effects of cell and substrate concentrations on d-allulose production from d-fructose by permeabilized C. glutamicum cells expressing DAEase from F. plautii.
(A) Effect of cell concentration. The reactions were performed by varying the cell concentration from 1 to 15 g/L in 50 mM PIPES buffer (pH 7.5) containing 750 g/L d-fructose in the presence of 1 mM Co2+ at 65°C for 10 min. (B) Effect of substrate concentration. The reactions were performed by varying the d-fructose concentration from 50 to 750 g/L in 50 mM PIPES buffer (pH 7.5) containing 10 g/L permeabilized cells in the presence of 1 mM Co2+ at 65°C for 10 min. Data represent the means of three separate experiments and error bars represent the standard deviation.
Fig 9.
Time-course reactions for the production of d-allulose from d-fructose by permeabilized and nonpermeabilized cells of C. glutamicum expressing DAEase from F. plautii.
The production of d-allulose (filled square) from d-fructose (filled circle) by permeabilized cells and the production of d-allulose (empty square) from d-fructose (empty circle) by nonpermeabilized cells. The reactions were performed in 50 mM PIPES buffer (pH 7.5) containing 10 g/L cells and 750 g/L d-fructose in the presence of 1 mM Co2+ at 65°C for 1 h. Data represent the means of three separate experiments and error bars represent the standard deviation.
Table 3.
d-Allulose production from d-fructose by whole recombinant cells expressing DAEase.