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The aim of the present work was to scale-up the protein production with enhanced proteolytic activity by phosphate addition in Jacaratia mexicana cell culture, by using the shear rate as scale-up criterion. The volumetric proteolytic activity in the J. mexicana cell culture in shake flask increased 1.9 times with increasing phosphate concentration from 1.25 to 2.5 mmol L-1, while the protein concentration was similar (similar to 55 mg L-1). This culture was scaled-up from 0.4-L to 4-L in stirred tank bioreactors at average shear rate of 200 s(-1) and 234 s(-1), respectively, increasing the phosphate concentration in the 4-L bioreactor (2.5 mmol L-1) with respect to the 0.4-L bioreactor (1.25 mmol L-1). There was also an increase in the volumetric proteolytic activity (2 times), without statistically significant difference in the protein concentration. Scale-up strategy and improvement of the culture medium exhibited a similar behaviour maintaining the protein productivity and increasing the proteolytic activity. These results indicate that the average shear rate is a useful hydrodynamic parameter in the scale-up of protein production of J. mexicana cell culture. Additional phosphate and bioreactor hydrodynamics could be related to unusual lengthening and thinning of most cells in the 4-L stirred tank bioreactor. Finally, the power input per unit volume in the scaled-up bioreactor decreased by 53%, improving the volumetric proteolytic activity and maintaining protein productivity. 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