Downstream strategy to obtain chymotrypsin from bovine pancreas using precipitation with an anionic strong polyelectrolyte.

BOERIS, VALERIA; ROMANINI, DIANA; FARRUGGIA, BEATRIZ; PICÓ, GUILLERMO

Recife

Workshop; 1º Workshop internacional en Biotecnología, III Encuentro Valnatura, III Jornada Científica do Lika; 2008

Valnarura

DOWNSTREAM STRATEGY TO OBTAIN CHYMOTRYPSIN FROM BOVINE PANCREAS USING PRECIPITATION WITH AN ANIONIC STRONG POLYELECTROLYTE. Valeria Boeris, Diana Romanini, Beatriz Farruggia and Guillermo A. Picó.                         Lab. de Biosepracion. Facultad de Ciencias Bioquímicas y Farmacéuticas. CONICET, FonCyT and CIUNR. Universidad Nacional de Rosario  Suipacha 570 (S2002RLK) Rosario. Argentina Email: pico@ifise.gov.ar   1. Introduction A great amounts of enzymes is required for different industrial proposes which makes necessary the develop of scaling up methodologies. In the zone where is located our laboratory, the meat industries are very important therefore, great amounts of wastes from these are produced. In this work was analyzed a method for isolation and purification of chymotrypsin from bovine pancreas homogenate by formation of an insoluble complex between the enzyme and a synthetic strong negatively charge polyelectrolyte such as the sodium salt of poly vinyl sulfonate (PVS) [1].   2. Matherials and Methods. Chymotrypsin (ChTRP) enzymatic activity: this assay is based on the hydrolysis of N-benzoyl-l-tyrosine ethyl ester (BTEE) [2].   ChTRP turbidimetric titration curves with polymer: The formation of the insoluble polymer-protein complex was followed by means of turbidimetric titration [2,3]. Bovine pancreas homogenate preparation: Bovine pancreas was homogenized  for 5 min. in a Minipimmer homogeneizator at a rate of 4000 rpm inmediatly processed. Zymogene activation from the bovine pancreas homogenate: ChTRP is present in the pancreas in a biological inactive form, therefore, it was activated by the presence of small concentration of trypsin (0.01 mg / g of homogenate) at pH 7.0 and the carried out at pH 2.5.   3. Results. 3.1. Titration of ChTRP with PVS: Fig. 1 shows the absorbance dependence at 420 nm when ChTRP at a constant concentration is titrated with PVS in media of pH 2.5. This pH was selected because ChTRP is stable a this pH. Also shows the residual activity in the supernatant solution before the precipitate was separed. It can be seen a decreases in the activity in the sence that the precipitate in formed confirming that PVS form a non soluble complex with ChTRP. The steoichiometric protein/polymer ratio which corresponds to the situation where the most protein has been precipitated as insoluble complex was calculated from Fig. 1. The ratio protein-polymer value found is important because it allows to calculate the minimal polymer necessary concentration to precipitate the enzyme.   3.3. ChTRP precipitation from the activated homogenate of bovine pancreas: aliquots with a mass of 10g of activated homogenate were titrated with increasing concentration of PVS solution (10 to 400 nM) by adding small aliquots of PVS concentrated solution. The precipitated obtained was separed by centrifugation and then dissolved by addition of 4 ml citrate-Tris-HCl buffer, pH 8.2. The ChTRP activity recovered and total protein concentration was determined in the supernatant. Fig. 2A-B shows that increasing in the PVS total concentration favours the ChTRP recovery reaching a maximum with a yielded of 61%, around a PVS concentration of 250 mM with purification factor ith values between 4.5 to 5.1. The presence of PEI induced a lost of the enzyme recovery.   4. Conclusion    Ours finding suggests that the prototropic groups of ChTRP lost their positive electrical change around 6, therefore the pH increase in this way induced a solubilization of the precipitate [4]. The low purification factor and recovery of ChTRP observed in the PEI presence is due to the interaction between the cationic polymer and nucleid acid with the anionic PVS. However, our finding suggests that the presence of nucleic acid in the homogenate does not induce any problem in the handly of the solution and in the protein precipitation and recovery. The 61 % of ChTRP recovered could be not be able increase due to the lost of the enzyme activity that occur when ChTRP is in the precipitate form at acid pH.  This cause should be due to the strong interaction of PVS with the positive charge groups of ChTRP, which should induces an irreversible conformational change in the tertiary structure of ChTRP near its catality site resulting in a partially  lost of the  enzyme biological activity. The presence of molecules of other protein (impurites) which they are included between the PVS-ChTRP complex molecules together in the moment of form the precipitate favours a decreasing in the enzyme purity.    Acknowledgements This work was supported by a grant from FoNCyT No. 06-12476/02 and CONICET PIP5053.   5. References 1. T. Matsudo, K. Ogawa, and E. Kokufuta.  Biomacromol.  4, 1794 (2003) 2. D. Romanini, M. Braia, R. Giatte Angarten, W. Loh and G. Picó. J.  Chromatography B,  25, 857 ( 2007) 3. V. A. Izumrudov, I. Y. Galaev, B. Mattiasson.  Bioseparation 7,  207 (1999) 4. V. Boeris, B. Farruggia, B. Nerli, D. Romanini, G. Picó. I. J. Biol. Macromol. 41, 286 (2007)