A sustainable affinity partitioning process to recover papain from Carica papaya latex using alginate as macro-ligand

• Hydrodynamic properties of alginate are modified by papain presence.
• Papain neutralizes alginate intra-chain charges and decreases the polymer stiffness.
• The alginate-papain interaction is cooperative and enthalpy-entropically driven.
• Affinity partitioning allows recovering papain from C. papaya latex sustainably.
• The proposed strategy uses a biodegradable salt and favors polymer-phase recycling.

The role of the natural biodegradable polymer alginate (ALG) as affinity macro-ligand for papain (PAP) was evaluated in order to design a new sustainable two-phase affinity strategy for recovering this enzyme from Carica papaya latex.In presence of PAP, decreased values of intrinsic viscosity and hydrodynamic radius of ALG were observed. These results suggested a neutralizing effect of PAP on the polymer negative charges which caused a decrease of intra-chain repulsion forces in ALG molecules and a consequent shrinkage of the polymer size. Calorimetric measurements demonstrated a cooperative interaction between PAP and ALG which was enthalpy-entropically driven.When partitioned C. papaya latex into aqueous two-phase systems (ATPSs) formed by polyethylene glycol (PEG) of MW 8000 and the biodegradable salt sodium citrate pH 5.20, only 20% of PAP was recovered at the PEG-enriched phase with a purification factor (PF) of 2.48. The addition of ALG 0.1% (w/w) into the system doubled the PAP partition coefficient (Kp, PAP), showing the ability of this polymer to enhance the enzyme recovery at the PEG-enriched phase and therefore, increasing the extraction efficiency. The subsequent addition of calcium chloride at a final concentration of 80 mM allowed the precipitation of the target enzyme and the recovering of PEG phase for recycling. The overall process showed a PAP recovery of 72% and a PF of 2.41. The proposed strategy not only conserved all the advantages of the reported extractions with PEG/ammonium sulfate (or phosphate) ATPSs, i.e., low cost, scalability and integration of the clarification and extraction steps, but also allowed overcoming their main drawbacks, i.e., the separation of the target molecule from the phase-polymer and the environmental impact caused by the disposal of these salts.