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Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte

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Abstract

The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity.

Introduction

Precipitation is a common approach to obtain enzymes and other macromolecules. This technique offers the possibility of concentrating and purifying the target macromolecule at a low cost. Polyelectrolyte precipitation uses a poly-charged macromolecule of opposite electrical charge to the target macromolecule, forming a soluble protein–polyelectrolyte complex. Under desired experimental conditions, these complexes interact among each other, producing insoluble macroaggregates. This is a suitable method for protein isolation because very low polyelectrolyte concentrations are used (up to 0.1%, w/w). This method sometimes offers a high selectivity and the insoluble complex can be re-dissolved by a pH change or adding a salt [1].

Production of proteins is a prime biotechnological application which includes upstream and downstream processing steps to obtain the final product in the desired purified form, the downstream processing being often the most expensive one. Bioseparation steps for the recovery of the final product can account for 50–80% of overall production costs. Most purification technologies use precipitation of proteins as one of the initial operations aimed at concentrating the product for further downstream steps. Precipitation using salts, organic solvents, non-ionic polymers and polyelectrolytes is a well known and simple technique for protein concentration. Attempts are usually made to derive some degree of purification of target products in the precipitation step [2].

One protease widely used in food and pharmaceutical industry is chymotrypsin which has a single polypeptidic chain of 324 amino acid residues and a molecular weight of 25.7 kDa. It is one of the proteolytic enzymes of vertebrate pancreas juice, being its optimum activity pH 8.2 and its isoelectric point 9.1 [3]. A great amount of this enzyme is required for different industrial purposes, which makes it necessary to develop scaling up methodologies. In the area where our laboratory is located meat industries are very important, therefore, great amounts of meat waste are produced. One of these products is the bovine pancreas, which is very rich in enzymes such as different types of protease, amylase and lipase of wide application in numerous biotechnological processes. In a previous paper [4] we described the molecular mechanism of interaction between polyvinil sulfonate (CH2–CH–O–SO32−)x, a strong anionic polyelectrolyte (pKa ≈ 1, molecular mass: 220 kDa) and determined the medium variable values at which the insoluble protein–polyelectrolyte formation is optimal. In this paper, these conditions are used as a method designed for the isolation and purification of this enzyme from bovine pancreas homogenate.

Section snippets

Chemicals

Chymotrypsin (ChTRP) and N-benzoyl-l-tyrosine ethyl ester (BTEE) were purchased from sigma Chem. Co., polyvinyl sulfonate (PVS), sodium salt in aqueous solution at 25% (w/w), from Aldrich and used without further purification. Buffer solutions formed by a mixture of sodium citrate (50 mM) and Tris–HCl (50 mM) of different pH were prepared, the pH was adjusted with NaOH or HCl in each case.

Bovine pancreas homogenate preparation

The pancreas was removed from a recently killed bovine, washed with isotonical saline solution and cut in

Titration of ChTRP with PVS

Fig. 1 shows the absorbance dependence at 420 nm when ChTRP is titrated at a constant concentration with PVS in pH 2.5 media. This pH was selected because ChTRP is stable at such pH, lower pH values were not assayed due to the possibility of enzyme denaturalization. A higher pH was not assayed because ChTRP forms aggregates and acquires biological activity from pH 6, inducing the auto-proteolysis of the enzyme. Fig. 1 also shows the residual activity in the supernatant solution before the

Discussion

There is a great interest in scaling up method for enzyme obtention using synthetic [4] and natural polyelectrolytes [9]. Chymotrypsinogen is activated to chymotrypsin by porcine enterokinase and the final step in the purification process is an ion exchange chromatography using NaCl gradient. All these operations are time-consuming, and cause a loss in the enzyme activity, thus making the standard methods expensive and difficult to scale up.

Below pH 6.0, ChTRP interacts with the anionic polymer

Acknowledgements

This work was supported by a grant from FoNCyT PICT06-12476/02 and CONICET PIP5053. We thank María Robson, Geraldine Raimundo, Mariana De Sanctis and Marcela Culasso for the language correction of the manuscript.

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