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.