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KMID : 1094720120170010076
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Biotechnology and Bioprocess Engineering
2012 Volume.17 No. 1 p.76 ~ p.83
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A new methanol-feeding strategy for the improved production of β-galactosidase in high cell-density fed-batch cultures of Pichia pastoris Mut+ strains
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Maghsoudi Amir
Hosseini Safoura
Shojaosadati Seyed Abbas
Vasheghani-Farahani Ebrahim
Nosrati Mohsen
Bahrami Ali
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Abstract
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Developing novel methanol-feeding strategies for the improved production of heterologous proteins in high cell-density fed-batch cultures of Pichia pastoris has been of great interest during recent years. In this study, a recombinant P. pastoris strain (GS115/His+ Mut+) producing β-galactosidase (β-Gal) was used to investigate conventional feeding strategies and to develop a new strategy to increase the recombinant protein production during fedbatch cultures on methanol. Three types of conventional methanol-feeding strategies, including μ-stat, dissolved oxygen-stat (DO-stat) and constant methanol concentration were investigated and compared with respect to alcohol oxidase (AOX), formate dehydrogenase (FDH) and β-gal activities, and cell dry weight (CDW), methanol, and formaldehyde concentration variations during the production phase. Methanol feeding with μ-stat 0.025/h exhibited the highest β-gal activity. Supplementing ammonium and magnesium in μ-stat 0.025/h did not affect the cell growth or methanol or formaldehyde concentrations throughout the fermentation but did improved the maximum β-gal activity from 148.2 to 158.1 kU/mL. A new three-step methanol-feeding strategy was developed based on the results obtained from conventional feeding strategies, which started with μ-stat 0.025/h for 5 h, then μ-stat 0.030/h, and finally, was switched to DO-stat when maintaining the DO above 20% air saturation became difficult. Implementation of this new feeding strategy resulted in a CDW of 107.2 ± 0.7 g/L, AOX specific activity of 0.1890 ± 0.0030 U/mg CDW, and β-gal activity of 173.5 ± 2.1 kU/mL after 29 h of fermentation, which shows a 5.6, 29.1, and 15.7% increase in CDW, AOX, and β-gal activity, respectively, compared to that of μ-stat at 0.025/h.
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KEYWORD
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Pichia pastoris, alcohol oxidase, methanol feeding, β-galactosidase, fed-batch fermentation, high cell-density
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