Production in stirred-tank bioreactor of recombinant bovine chymosin B by a high-level expression transformant clone of "Pichia pastoris"

BOZZO, J.; RECUPERO, M.N.; GALVAGNO, M.A.; NOSEDA, D.; BLASCO, M.

PROTEIN EXPRESSION AND PURIFICATION

ACADEMIC PRESS INC ELSEVIER SCIENCE

Lugar: Amsterdam; Año: 2016 vol. 123 p. 112 - 121

1046-5928

An intense screening of Pichia pastoris clones transformed with the gene of bovine chymosin undermethanol-inducible AOX1 promoter was performed, obtaining a transformant clone with a higher milkclottingactivity value in comparison with our previous studies. The scaling of recombinant-chymosinproduction was carried out by a fed-batch strategy in a stirred-tank bioreactor using biodieselbyproductcrude glycerol as the carbon source and pure methanol for the induction of chymosinexpression, achieving a biomass concentration of 158 g DCW/L and a maximum coagulant activity of 192IMCU/ml after 120 h of methanol induction. Recombinant bovine chymosin was purified from bioreactorfermentationculture by a procedure including anion-exchange chromatography which allowedobtaining heterologous chymosin with high level of purity and activity; suggesting that this downstreamstep could be scaled up in a successful manner for chymosin purification. Thermoestability assaypermitted to establish that unformulated recombinant chymosin could be stored at 5 C withoutdecrease of enzyme activity throughout at least 120 days. Finally, reiterative methanol-inductions ofrecombinant chymosin expression in bioreactor demonstrated that the reutilization of cell biomassovercame the low enzyme productivity usually reached by P. pastoris system.