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

• We performed a screening for Pichia pastoris clones transformed with bovine prochymosin gene with high expression levels of recombinant chymosin and therefore high milk-clotting activity.
• The production of recombinant bovine chymosin by a high-producer clone was scaled up in a stirred-tank bioreactor using fed-batch methanol feeding under optimized conditions.
• Biodiesel-byproduct crude glycerol was used as a low cost of carbon source, which reduces the process cost for the production of recombinant bovine chymosin.
• Recombinant bovine chymosin was purified from bioreactor-fermentation culture by anion-exchange chromatography obtaining heterologous chymosin with high level of purity.
• Thermoestability assay permitted to establish that unformulated recombinant chymosin could be stored at 5 °C without decrease of enzyme activity during 120 days.
• Reiterative methanol-inductions of recombinant chymosin expression demonstrated that the reutilization of P. pastoris biomass increases heterologous enzyme productivity.

An intense screening of Pichia pastoris clones transformed with the gene of bovine chymosin under methanol-inducible AOX1 promoter was performed, obtaining a transformant clone with a higher milk-clotting activity value in comparison with our previous studies. The scaling of recombinant-chymosin production was carried out by a fed-batch strategy in a stirred-tank bioreactor using biodiesel-byproduct crude glycerol as the carbon source and pure methanol for the induction of chymosin expression, achieving a biomass concentration of 158 g DCW/L and a maximum coagulant activity of 192 IMCU/ml after 120 h of methanol induction. Recombinant bovine chymosin was purified from bioreactor-fermentation culture by a procedure including anion-exchange chromatography which allowed obtaining heterologous chymosin with high level of purity and activity; suggesting that this downstream step could be scaled up in a successful manner for chymosin purification. Thermoestability assay permitted to establish that unformulated recombinant chymosin could be stored at 5 °C without decrease of enzyme activity throughout at least 120 days. Finally, reiterative methanol-inductions of recombinant chymosin expression in bioreactor demonstrated that the reutilization of cell biomass overcame the low enzyme productivity usually reached by P. pastoris system.