Bovine lactoferrin (bLf) has been well characterized as a multifunctional glycoprotein belonging to the transferrin family with antibacterial, antioxidant, anti-tumor, and antiviral activities. The main aim of this study was to determine the feeding strategies in fed-batch fermentation to obtain high-cell density in Pichia pastoris KM71H-3 for the production of bLf. During the growth phase, two feeding strategies were performed: (i) Feeding glycerol solution; and (ii) feeding glycerol and nitrogen source solution with a carbon-to-nitrogen ratio of 3:1. In addition, two feeding induction strategies were applied: One-time feeding and continuous feeding with 0.5% of methanol every 24 h. The results showed that the highest cell density at OD600 was 362.67 ± 2.04 when the feeding phase used a mix-feed solution of glycerol and nitrogen sources with a carbon to nitrogen ratio of 3:1 in the 2-L scale. Up-scaled production of lactoferrin from this strain was successfully employed in the 10-L and 100-L bioreactor with the highest OD600 reaching 338.20 ± 3.38 and 375.50 ± 2.98, respectively. For optimal lactoferrin expression, methanol was fed continuously, corresponding to an induced methanol concentration of 0.5% per 24 h. The appropriate induction time was 48 h. This research provides information on cell growth and fed-batch strategies for enhanced bLf production using P. pastoris as a host, which may be applicable to the expression of other proteins from P. pastoris strains.
Key words: lactoferrin, Pichia pastoris, fed-batch fermentation, high cell density fermentation strategy
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