NERLI Bibiana Beatriz
congresos y reuniones científicas
An Improving Method for Purifying a Protease of High Commercial Value, the Bovine Pancreatic Trypsin
Saltillo, Coahuila. MEXICO
Congreso; 2nd. Int. Congress: "Food Science & Food Biotechnology in Developing Countries"; 2006
Institución organizadora:
Mexican Association of Food Science
Bovine trypsin (TRP) is employed as a dietary supplement in the food industry and in substitutive therapies for patients with pancreatic diseases. Traditional methods isolate TRP from pancreas where it is stored as its inactive precursor, trypsinogen (TRPz). Alpha-chymotrypsin (ChTRP), also stored as alpha-chymotrypsinogen (ChTRPz), represents the principal trypsin contaminant since both enzymes exhibit chemical similarities.  During the purification process, the zymogens are activated and then separated each other and from other proteases through chromatographic steps which require expensive materials. Recently, the liquid-liquid extraction principle applied to aqueous two-phase systems (ATPSs) have been successfully used for purifying industrial proteins due to its ease scaling-up. The most commonly used ATPS is formed by  polyethyleneglycol (PEG) and potassium phosphate but the high salt concentration required represents an environmental problem. The goal of this study is to choose the most adequate conditions for separating TRP from ChTRP and TRPz from ChTRPz (without activation process) by employing PEG/sodium citrate (NaCit) ATPSs since citrate is a biodegradable and non toxic anion. We analysed the effect of polymer molecular weight (MW), pH and tie line length on the partition coefficient of the assayed proteins. TRP/ChTRP mixtures showed different separating conditions from those formed by their zymogens (TRPz/ChTRPz). ATPSs formed by PEG of MW 3,350 and NaCit pH 5.2 showed to have the best capability of separating TRP from ChTRP. About 20 % of TRP was recovered in the top phase with a purity of 62 %.  The addition of NaCl 3 % to the ATPSs increased both the recovery and the purity to 65 % and 77 % respectively. On the other hand, ATPSs formed by PEG of MW 1,450 and NaCit pH 8.2 allowed to separate TRPz from ChTRPz, recovering 38 % of TRPz in the bottom phase with a purity of 66 %. High top-bottom volume ratios slightly decreased the recovery (30 %) but increased the purity to 80 %. The obtained  information can be useful as a starting point to be applied to the TRP isolation from bovine pancreas, which represent an abundant and inexpensive subproduct of the meat industry in our country. keywords[trypsin, chymotrypsin, aqueous two-phase systems, partitioning]