INVESTIGADORES
CAMPERI Silvia Andrea
congresos y reuniones científicas
Título:
MULTI-STEP PARTITIONING AND PARTIAL PURIFICATION OF TRYPSIN IN AQUEOUS TWO-PHASE SYSTEMS
Autor/es:
MARIELA M. MARANI; SILVIA A. CAMPERI; OSVALDO CASCONE
Lugar:
San Miguel de Tucumán, Argentina
Reunión:
Simposio; Simposio Internacional de Biotecnología Simposio Argentino-Italiano de Bacterial Lácticas; 2004
Resumen:
 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. Trypsin is a proteolytic enzyme usually employed in food, pharmaceutical and leather industries as well as in biochemical research. The influence of PEG molecular weight, pH and NaCl on the partition coefficient of trypsin  (Ktry) and total protein (Ktp) of a pancreatic extract was assessed. NaCl addition to 1 molal increased dramatically Ktry from 0.25 to 14.62. On the basis of this study, a multi-step purification process was designed: the pancreas crude extract was added to a 15 % PEG 10,000 - 8.8 % MgSO4 ATPS. The bottom phase was re-extracted once with fresh top phase, then NaCl was added to transfer the trypsin to the top phase and in the last step the top phase was re-extracted with fresh bottom phase. The purification factor achieved was 8.4, with a yield of 75 %. In order to understand the partitioning behaviour of the accompanying proteins, a trypsin-free crude extract was used. The contaminants partition coefficients (Kcp) on the succesive steps were: Kcp1=0.08, Kcp2=0.07, Kcp3=1.34, Kcp4=14.15      
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