PARTITION BEHAVIOR AND PARTIAL PURIFICATION OF TRYPSIN IN AQUEOUS TWO-PHASE POLY(ETHYLENEGLYCOL) AND MAGNESIUM SULFATE SYSTEMS

MARANI, MARIELA; CAMPERI, SILVIA; NUSBLAT, ALEJANDRO; CASCONE, OSVALDO

Santiago de Chile, Chile

Congreso; 12 th International biotechnology symposium and exhibition; 2004

Conicyt - IUPAC

Aqueous two-phase system (ATPS) is an integrative purification process that combines clarification and coarse purification in one step, thus reducing the downstream processing costs. This extraction technology offers high capacity, high activity yields and easy scale up. Polymer-salt systems have the advantage of low viscosity and low cost compared to polymer-polymer systems. To resolve complex mixtures of proteins, e.g. from cell extracts, single-step partitioning is often inadequate, in such cases the use of multistep extraction procedures is indicated. Trypsin is a proteolytic enzyme usually employed in pharmaceutical, food and leather industries as well as in biochemical research. Trypsin behavior in poly(ethyleneglycol) (PEG) and magnesium sulfate ATPSs was characterized in order to find a suitable system for its purification. Phase diagrams of PEG-magnesium sulfate for different PEG MW were determined. The influence of PEG MW, pH and NaCl on the partition coefficient of trypsin (KTRY) and total protein (KTP) of a pancreatic extract was assessed. In the systems studied, trypsin partition was only slightly influenced by tie-line length and did not show any regular trend with pH. The KTRY decreased from 0.4 to 0.15 and the KTP from 0.85 to 0.47 by increasing the PEG MW from 4000 to 10000. The NaCl addition changed dramatically the KTRY from 0.15 to 23 but no significant effect on KTP was evidenced. If the enzyme is retained in the upper phase by an adequate amount of NaCl, it is posible to improve the removal of contaminating proteins by using multistep extraction procedures. Systems studied were: A) PEG 15%/MgSO4 8.8%/1M NaCl: after the initial partitioning  the top phase was re-extracted three times with fresh bottom phase. B) PEG 15%/MgSO4 8.8%: in this case the bottom phase was re-extracted two times with fresh top phase and then NaCl  was added to transfer the trypsin to the top phase and C) PEG 15%/MgSO4 8.8%: in this case the bottom phase was re-extracted once with fresh top phase and then NaCl was added to transfer the trypsin to the top phase. In the last step, the top phase was re-extracted with fresh bottom phase. The purification factors achieved were: A) 5.5; B) 2.6; C) 8.4. These results may be considered as an interesting alternative for the use of ATPS in trypsin purification from crude extracts.