continuous isolation of trypsin by affinity adsorbent recycling

• Published in: Biotechnology and bioengineering 1997-12, Vol.56 (5), p.538-545
• Main Authors: Gill, A.L, Niven, G.W, Scurlock, P.G
• Format: Article
• Language: English
• Subjects: Adsorbents; affinity; Amino acids; Biological and medical sciences; Biological Sciences; Biotechnology; continuous; Enzyme engineering; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Methods. Procedures. Technologies; protein; Proteins; Purification; Recycling; Separation; trypsin
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(19971205)56:5<538::AID-BIT7>3.0.CO;2-J

A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 micrograms of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%.