Fermentation optimization for production of lactoferrin N-lobe by recombinant Bacillus subtilis

Abstract:

To achieve an efficient preparation of lactoferrin N-lobe, we optimized the fermentation process for a recombinant Bacillus subtilis pMA0911-D60Y/Y92D producing lactoferrin N-lobe. The IOD of the lactoferrin N-lobe reached 68.03% under the optimized cultural conditions, that is using glucose and tryptone as the best carbon and nitrogen source, respectively, and conduct the fermentation under pH 7.0, 28 ℃, for 25.5 h. An optimized fermentation process was obtained through fermentation optimization on a 10 L fermenter. That is, culturing the recombinant strain at 30 ℃, pH 7.5 within 0-7 h, and switching to induction at 28 ℃, pH 7.5 within 7-25 h for production of lactoferrin N-lobe,using an agitation speed of 300 r/min throughout the fermentation. After the fermentation, the cells were collected and disrupted, followed by purification of the lactoferrin N-lobe to homogeneity by using HisTrap HP-affinity and a Superdex^TM 200(10/300 GL)-affinity chromatography. The purified lactoferrin N-lobe proteins with over 94% purity were obtained. One liter culture of recombinant B. subtilis pMA0911-D60Y/Y92D produced 23.5 mg of pure protein. This study may facilitate the fermentative production of the recombinant lactoferrin N-lobe.

Key words:bovine lactoferrin N-lobe;Bacillus subtilis;protein expression and purification;fermentation process optimization;response surface analysis