Papain-Poly (vinyl sulfonate) Complex Formation as the First Stage in Bioseparation

BRAIA, MAURICIO; PORFIRI, MARIA CECILIA; FERRERO, MAXIMILIANO; ROCHA, MARIA VICTORIA; TUBIO, GISELA; ROMANINI, DIANA

Puerto Vallarta

Congreso; 16th International Conference on Biopartitioning and Purification BPP 2011; 2011

The latex of Carica papaya is a rich source of cysteine endopeptidases, including papain, glycyl endopeptidase, chymopapain and caricain, which constitute more than 80% of the whole enzyme fraction. Papain (EC 3.4.22.2) is a minor constituent (5-8%) among the papaya endopeptidases. The enzyme is used widely as meat tenderizer, and has also several other applications, e.g. for defibrinating wounds, treatment of edemas, shrink proofing of wool, etc.Purification of papain from papaya latex has traditionally been achieved by precipitation methods; however, the purified enzyme still remains contaminated with other proteases. Improving bioseparation requires the use of various chromatographic techniques including ion exchange, covalent, or affinity chromatography.When an insoluble polymer-protein complex is specifically formed with one of the proteins in a crude extract followed by a phase separation, the process can be used as a convenient strategy for the isolation and purification of a target protein. Precipitation as a product-concentration step offers several advantages in that it is easy to scale up, uses simple equipment and can be based on a large variety of alternative precipitants.The removal of the polymer requires an experimental procedure such as molecular exclusion chromatography, ultrafiltration, etc. However, this last step depends on the subsequent application of the protein. Papain is involved, for example, in the process of leather softening. In this case, there is no need to remove the polymer.This work reports investigation aiming at verifying the existence of specific interactions and insoluble complex formation between papain from Carica papaya and poly (vinyl sulfonate) using spectroscopy techniques. The papain-poly(vinyl sulfonate) complex was insoluble at pH lower than 6, with a polymer/protein molar ratio of 1/279. The presence of low ionic strength (0.25M) can easily redissolve the complex. This suggests that electrostatic forces are involved in the interaction between the protein and the polymer.Papain activity in the presence of poly (vinyl sulfonate) is higher than in its absence and the variation of the activity through time is lower in the presence of the polymer. In this way, poly (vinyl sulfonate) protects the enzyme from itself degradation and makes it more stable.The recovery obtained after PAP precipitation with PVS from latex was high (86.98 %) which prove that the methodology is suitable for concentrating the enzyme or as a first extractive step in the purification designs. However, the purification factor obtained was low (2.08). We are working to find the conditions that can improve the purification factor.Authors would like to thank FONCyT (PICT-2008-0186) for financial support.