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Abstract: The combination of two bioseparation techniques, partition in aqueous two-phase systems and polyelectrolyte precipitation of the target enzyme from the phase where it is present, was assayed to purify pepsin from bovine abomasum homogenate. Pepsin was partitioned in favor of the polyethyleneglycol-rich phase in an aqueous two-phase system of polyethyleneglycol 600 and 1450-sodium phosphate; however, a great amount of impure proteins were present. Chitosan (a cationic natural polyelectrolyte) was added to precipitate this acid enzyme as a form of insoluble complex. The addition of this second step increased the purity of the enzyme significantly while the yield was not significantly decreased. The combination of both partition in polyethyleneglycol 1450-phosphate system and chitosan precipitation produced a pepsin recovery of 48.5% with a purification factor of 9.0. The biological activity of the recovered enzyme remained unaltered. [Copyright &y& Elsevier]

Abstract: The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity. [Copyright &y& Elsevier]

Abstract: The separation of chymotrypsin from a crude filtrate of fresh bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged natural strong polyelectrolyte: ι-carrageenan. The zymogen form of the enzyme was activated by addition of trypsin (0.0001%, w/w), then, the enzyme was precipitated by polyelectrolyte addition at pH 4.50. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.20. The recovery of chymotrypsin biological activity was 60% of the initial activity in the homogenate with 3-fold increase in its specific activity. The volume of the final product decreased to 10% of the feedstock, concentrating the sample up to 10 times. [Copyright &y& Elsevier]