Multi-step partitioning and partial purification of trypsin in aqueous two-phase systems

MARANI MARIELA M.; CAMPERI SILVIA A.; CASCONE OSVALDO

San Miguel de Tucumán, Tucumán, ARGENTINA

Simposio; SIB Simposio Internacional de Biotecnología; 2004

CONICET-Agregaduría Científica Embajada de Italia-Agencia Nacional de Promoción Científica y Tecnológica

Recovery and purification processes account for up to 80 % of the production cost of a protein of biotechnological interest. There is a need for efficient and economic large-scale bioseparation techniques. Aqueous two-phase systems (ATPS) allow process integration as simultaneous separation and concentration of the target protein is achieved. The low cost and easy scale-up allows its potential use in large-scale downstream processing of proteins. Moreover, the ATPS biocompatible environment facilitates the preservation of the biological activity of enzyme extracts. The use of polymer-salt ATPS has the advantage of low viscosity and low cost compared to polymer-polymer ATPS. To resolve complex mixtures of proteins such as cell extracts, a single-step partitioning is often inadequate, and the use of multi-step fractionation procedures is indicated.  Trypsin is a proteolytic enzyme usually employed in food, pharmaceutical and leather industries as well as in biochemical research. In order to find a suitable ATPS for trypsin purification from a crude pancreas extract, the multi-step partition behaviour in a PEG-magnesium sulphate ATPS was studied. The influence of PEG molecular weight, pH and NaCl on the partition coefficient of trypsin (KTRY) and total protein (KTP) of the pancreatic extract was assessed. The NaCl addition to 1 molal increased dramatically KTRY from 0.15 to 23. If the enzyme is retained in the top phase by an adequate concentration of NaCl, it is possible to improve the removal of contaminating proteins by using multi-step extraction procedures. Systems studied were: a) PEG 15 % - MgSO4 8.8 % - 1 molal NaCl: after the initial partitioning the top phase was re-extracted 3 times with fresh bottom phase, b) PEG 15 % - MgSO4 8.8 %: the bottom phase was re-extracted twice with fresh top phase to remove contaminants, and then NaCl was added to transfer the trypsin to the top phase, c) PEG 15 % - MgSO4 8.8 %: in this case 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 factors achieved were: a) 5.5, b) 2.6, c) 8.4, thus evidencing that process c is the more appropriate for trypsin purification. In order to understand the partitioning behaviour of the accompanying proteins in this process, a trypsin-free crude extract was used. The contaminants partition coefficient (KCP) on the successive steps were: KCP1 = 0.077, KCP2 = 0.072, KCP3 =1.338, KCP4 = 14.15. To improve the performance of the process, a PEG derivative with an affinity ligand for trypsin will be synthesised.